Agonists of Guanylate Cyclase Useful For the Treatment of Gastrointestinal Disorders, Inflammation, Cancer and Other Disorders

ABSTRACT

The invention provides novel guanylate cyclase-C agonist peptides and their use in the treatment of human diseases including gastrointestinal disorders, inflammation or cancer (e.g., a gastrointestinal cancer). The peptides can be administered either alone or in combination with an inhibitor of cGMP-dependent phosphodiesterase. The gastrointestinal disorder may be classified as irritable bowel syndrome, constipation, or excessive acidity etc. The gastrointestinal disease may be classified as either inflammatory bowel disease or other GI condition, including Crohn&#39;s disease and ulcerative colitis, and cancer.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/010,267 filed on Jan. 20, 2011, which is a continuationapplication of U.S. application Ser. No. 12/133,344, now U.S. Pat. No.7,879,802, filed on Jun. 4, 2008, which claims the benefit of priorityto U.S. Provisional Application No. 60/933,194 filed on Jun. 4, 2007,the contents of each of which are incorporated by reference in theirentireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “40737-503C02US_ST25”, which wascreated on Apr. 3, 2013 and is 67 KB in size, are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the therapeutic use of guanylatecyclase C (GC-C) agonists as a means for enhancing the intracellularproduction of cGMP. The agonists may be used either alone or incombination with inhibitors of cGMP-specific phosphodiesterase toprevent or treat inflammation, cancer and other disorders, particularlyof the gastrointestinal tract and the lung.

BACKGROUND OF THE INVENTION

Uroguanylin, guanylin and bacterial ST peptides are structurally relatedpeptides that bind to a guanylate cyclase receptor and stimulateintracellular production of cyclic guanosine monophosphate (cGMP) (1-6).This results in the activation of the cystic fibrosis transmembraneconductance regulator (CFTR), an apical membrane channel for efflux ofchloride from enterocytes lining the intestinal tract (1-6). Activationof CFTR and the subsequent enhancement of transepithelial secretion ofchloride lead to stimulation of sodium and water secretion into theintestinal lumen. Therefore, by serving as paracrine regulators of CFTRactivity, cGMP receptor agonists regulate fluid and electrolytetransport in the GI tract (1-6; U.S. Pat. No. 5,489,670). Thus, thecGMP-mediated activation of CFTR and the downstream signaling plays animportant role in normal functioning of gut physiology. Therefore, anyabnormality in this process could potentially lead to gastrointestinaldisorders such as irritable bowel syndrome, inflammatory bowel disease,excessive acidity and cancer (25, 26).

The process of epithelial renewal involves the proliferation, migration,differentiation, senescence, and eventual loss of GI cells in the lumen(7, 8). The GI mucosa can be divided into three distinct zones based onthe proliferation index of epithelial cells. One of these zones, theproliferative zone, consists of undifferentiated stem cells responsiblefor providing a constant source of new cells. The stem cells migrateupward toward the lumen to which they are extruded. As they migrate, thecells lose their capacity to divide and become differentiated forcarrying out specialized functions of the GI mucosa (9). Renewal of GImucosa is very rapid with complete turnover occurring within a 24-48hour period (9). During this process mutated and unwanted cells arereplenished with new cells. Hence, homeostasis of the GI mucosa isregulated by continual maintenance of the balance between proliferationand apoptotic rates (8).

The rates of cell proliferation and apoptosis in the gut epithelium canbe increased or decreased in a wide variety of different circumstances,e.g., in response to physiological stimuli such as aging, inflammatorysignals, hormones, peptides, growth factors, chemicals and dietaryhabits. In addition, an enhanced proliferation rate is frequentlyassociated with a reduction in turnover time and an expansion of theproliferative zone (10). The proliferation index has been observed to bemuch higher in pathological cases of ulcerative colitis and other GIdisorders (11). Thus, intestinal hyperplasia is the major promoter ofgastrointestinal inflammation and carcinogenesis.

In addition to a role for uroguanylin and guanylin as modulators ofintestinal fluid and ion secretion, these peptides may also be involvedin the continual renewal of GI mucosa by maintaining the balance betweenproliferation and apoptosis in cells lining GI mucosa. Therefore, anydisruption in this renewal process, due to reduced production ofuroguanylin and/or guanylin can lead to GI inflammation and cancer (25,26). This is consistent with previously published data in WO 01/25266,which suggest a peptide with the active domain of uroguanylin mayfunction as an inhibitor of polyp development in the colon and mayconstitute a treatment of colon cancer. However, recent data alsosuggest that uroguanylin also binds to a currently unknown receptor,which is distinct from GC-C receptor (3,4). Knockout mice lacking thisguanylate cyclase receptor show resistance to ST peptides in theintestine, but effects of uroguanylin and ST peptides are not disturbedin the kidney in vivo (3). These results were further supported by thefact that membrane depolarization induced by guanylin was blocked bygenistein, a tyrosine kinase inhibitor, whereas hyperpolarizationinduced by uroguanylin was not effected (12, 13). Thus, it is not clearif the anti-colon cancer and anti-inflammatory activities of uroguanylinand its analogs are mediated through binding to one or both of thesereceptors.

Inflammatory bowel disease is a general name given to a group ofdisorders that cause intestines to become inflamed, characterized by redand swollen tissue. Gastrointestinal (GI) inflammation can be a chroniccondition and often leads to GI cancer (14). Examples of suchinflammatory bowel diseases (IBD) include Crohn's disease and ulcerativecolitis (UC). It is estimated that as many as 1,000,000 Americans areafflicted with IBD, with male and female patients appearing to beequally affected. Most cases are diagnosed before age 30, but thedisease can occur in the sixth, seventh, and later decades of life.

Crohn's disease is a serious inflammatory disease that predominantlyeffects ileum and colon, but can also occur in other sections of the GItract, whereas UC is exclusively an inflammatory disease of the colon,the large intestine (15). Unlike Crohn's disease, in which all layers ofthe intestine are involved, and in which there can be normal healthybowel in between patches of diseased bowel, UC affects only theinnermost lining (mucosa) of the colon in a continuous manner (16).Depending on which portion of the GI tract is involved, Crohn's diseasemay be referred to as ileitis, regional enteritis, colitis, etc. Crohn'sdisease and UC differ from spastic colon or irritable bowel syndrome,which are motility disorders of the GI tract.

While the precise cause of IBD is not known, it is believed that thedisruption of the process of continual renewal of GI mucosa may beinvolved in disease (17,18). The renewal process of the GI lining is anefficient and dynamic process involving the continual proliferation andreplenishment of unwanted damaged cells. Proliferation rates of cellslining the GI mucosa are very high, second only to the hematopoieticsystem. Thus, the balance between proliferation and apoptosis isimportant to the maintenance of the homeostasis of the GI mucosa(19,20).

GI homeostasis depends on both proliferation and programmed cellulardeath (apoptosis) of epithelial cells lining the gut mucosa. Hence,cells are continually lost from the villus into the lumen of the gut andare replenished at a substantially equal rate by the proliferation ofcells in the crypts, followed by their upward movement to the villus. Ithas become increasingly apparent that the control of cell death is anequally, if not more, important regulator of cell number andproliferation index (19,20). Reduced rates of apoptosis are oftenassociated with abnormal growth, inflammation, and neoplastictransformation. Thus, both decreased proliferation and/or increased celldeath may reduce cell number, whereas increased proliferation and/orreduced cell death may increase the proliferation index of intestinaltissue (20), which may lead to GI inflammatory diseases and cancer.

Uroguanylin and guanylin peptides also appear to promote apoptosis bycontrolling cellular ion flux. Alterations in apoptosis have beenassociated with tumor progression to the metastatic phenotype. While aprimary gastrointestinal (GI) cancer is limited to the small intestine,colon, and rectum, it may metastasize and spread to such localities asbone, lymph nodes, liver, lung, peritoneum, ovaries, and brain. Byenhancing the efflux of K+ and influx of Ca++, uroguanylin and relatedpeptides may promote the death of transformed cells and thereby inhibitmetastasis

Irritable bowel syndrome (IBS) and chronic idiopathic constipation arepathological conditions that can cause a great deal of intestinaldiscomfort and distress but unlike the IBD diseases such as ulcerativecolitis and Crohn's disease, IBS does not cause the serious inflammationor changes in bowel tissue and it is not thought to increase the risk ofcolorectal cancer. In the past, inflammatory bowel disease (IBD), celiacdisease and irritable bowel syndrome (IBS) were regarded as completelyseparate disorders. Now, with the description of inflammation, albeitlow-grade, in IBS, and of symptom overlap between IBS and celiacdisease, this contention has come under question. Acute bacterialgastroenteritis is the strongest risk factor identified to date for thesubsequent development of postinfective irritable bowel syndrome.Clinical risk factors include prolonged acute illness and the absence ofvomiting. A genetically determined susceptibility to inflammatorystimuli may also be a risk factor for irritable bowel syndrome. Theunderlying pathophysiology indicates increased intestinal permeabilityand low-grade inflammation, as well as altered motility and visceralsensitivity (27). Serotonin (5-hydroxytryptamine [5-HT]) is a keymodulator of gut function and is known to play a major role inpathophysiology of IBS. It has been shown that the activity of 5-HT isregulated by cGMP (28). Therefore, based on this observation as well asother effects of cGMP, we believe that GC-C agonists will be useful inthe treatment of IBS.

Given the prevalence of inflammatory conditions in Western societies andthe attendant risk of developing cancerous lesions from inflamed tissue,particularly intestinal tissue, a need exists to improve the treatmentoptions for inflammatory conditions, particularly of thegastrointestinal tract.

SUMMARY OF THE INVENTION

The present invention is based upon the development of agonists ofguanylate cyclase receptor. The agonists are analogs of uroguanylin andbacterial ST peptides and have superior properties such as for examplehigh resistance to degradation at the N-terminus and C-terminus fromcarboxypeptidases and/or by other proteolytic enzymes present in thestimulated human intestinal juices and human gastric juices.

The peptides of the invention may be used to treat any condition thatresponds to enhanced intracellular levels of cGMP. Intracellular levelsof cGMP can be increased by enhancing intracellular production of cGMPand/or by inhibition of its degradation by cGMP-specificphosphodiesterases. Among the specific conditions that can be treated orprevented are gastrointestinal disorders, inflammatory disorders, lungdisorders, cancer, cardiac disorders, eye disorders, oral disorders,blood disorders, liver disorders, skin disorders, prostate disorders,endocrine disorders, increasing gastrointestinal motility and obesity.Gastointestinal disorders include for example, irritable bowel syndrome(IBS), non-ulcer dyspepsia, chronic intestinal pseudo-obstruction,functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux,gastroesophageal reflux disease (GERD), ileus inflammation (e.g.,post-operative ileus), gastroparesis, heartburn (high acidity in the GItract), constipation (e.g., constipation associated with use ofmedications such as opioids, osteoarthritis drugs, osteoporosis drugs;post surgical constipation, constipation associated with neuropathicdisorders. Inflammatory disorders include tissue and organ inflammationsuch as kidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); pancreaticinflammation (e.g., pancreatis), lung inflammation (e.g., bronchitis orasthma) or skin inflammation (e.g., psoriasis, eczema). Lung Disordersinclude for example chronic obstructive pulmonary disease (COPD), andfibrosis. Cancer includes tissue and organ carcinogenesis includingmetatases such as for example gastrointestinal cancer, (e.g., gastriccancer, esophageal cancer, pancreatic cancer colorectal cancer,intestinal cancer, anal cancer, liver cancer, gallbladder cancer, orcolon cancer; lung cancer; thyroid cancer; skin cancer (e.g., melanoma);oral cancer; urinary tract cancer (e.g. bladder cancer or kidneycancer); blood cancer (e.g. myeloma or leukemia) or prostate cancer.Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high tryglycerides. Liverdisorders include for example cirrhosis and fibrosis. In addition, GC-Cagonist may also be useful to facilitate liver regeneration in livertransplant patients. Eye disorders include for example increasedintra-ocular pressure, glaucoma, dry eyes retinal degeneration,disorders of tear glands or eye inflammation. Skin disorders include forexample xerosis. Oral disorders include for example dry mouth(xerostomia), Sjögren's syndrome, gum diseases (e.g., periodontaldisease), or salivary gland duct blockage or malfunction. Prostatedisorders include for example benign prostatic hyperplasia (BPH).Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

In one aspect, the present invention is directed to a peptide consistingessentially of the amino acid sequence of, SEQ ID NOs: 2-54 and 57-98and to therapeutic compositions which contain these peptides. Preferredpeptides include SEQ ID NO: 8, 9, 10, 58 and 59. The term “consistingessentially of” includes peptides that are identical to a recitedsequence identification number and other sequences that do not differsubstantially in terms of either structure or function. For the purposeof the present application, a peptide differs substantially if itsstructure varies by more than three amino acids from a peptide of SEQ IDNOs 2-54 and 57-98 or if its activation of cellular cGMP production isreduced by more than 50% compared to a control peptide such as SEQ IDNO:1, 55 or 56. Preferably, substantially similar peptides should differby no more than two amino acids and not differ by more than about 25%with respect to activating cGMP production. The instant peptidesequences comprise at least 12 amino acid residues, preferably between12 and 26 amino acids in length.

The peptides may be in a pharmaceutical composition in unit dose form,together with one or more pharmaceutically acceptable carrier,excipients or diluents. The term “unit dose form” refers to a singledrug delivery entity, e.g., a tablet, capsule, solution or inhalationformulation. The amount of peptide present should be sufficient to havea positive therapeutic effect when administered to a patient (typically,between 100 μg and 3 g). What constitutes a “positive therapeuticeffect” will depend upon the particular condition being treated and willinclude any significant improvement in a condition readily recognized byone of skill in the art. For example, it may constitute a reduction ininflammation, shrinkage of polyps or tumors, a reduction in metastaticlesions, etc.

In yet another aspect, an invention provides administering to saidpatient an effective dose of an inhibitor of cGMP-specificphosphodiesterase (cGMP-PDE) either concurrently or sequentially withsaid guanylate cyclase receptor agonist. The cGMP-PDE inhibitor includefor example suldinac sulfone, zaprinast, and motapizone, vardenifil, andsildenafil. In addition, GC-C agonist peptides may be used incombination with inhibitors of cyclic nucleotide transporters.

Optionally, anti-inflammatory agents are also administered.Anti-inflammatory agents include for example steroids and non-steroidalanti-inflammatory drugs (NSAIDS).

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar chart showing the biological activity of SP-304 afterincubation with simulated gastric fluid (SGF) for times as indicated.The biological activity of SP-304 was determined by measuring itsability to stimulate cGMP synthesis in T84 cells. Following theincubations, samples were used for their abilities to stimulate cGMPsynthesis in T84 cells. The cGMP stimulation activity in sample at 0 minof incubation with SGF was taken as 100%. The activities in samples fromother times of incubations with SGF were calculated as percentage of theactivity in the sample at 0 min. The data is average of triplicates±SD

FIG. 1B is a schematic representation of the results of HPLCchromatographic analyses of SP-304 samples after incubation with SGF atindicated times. The major peak of SP-304 did not change followingincubation with SGF, indicating that the peptide was resistant to SGFdigestion. The arrows indicate the elution position of SP-304.

FIG. 2A is a bar chart showing Cyclic GMP synthesis in T84 cells bySP304 samples after incubation with simulated intestinal fluid (SIF) forthe indicated times. Following the incubations, samples were used fortheir abilities to stimulate cGMP synthesis in T84 cells. The cGMPstimulation activity in sample at 0 min of incubation with SIF was takenas 100%. The activities in samples from other times of incubations withSIF were calculated as percentage of the activity in the sample at 0min. The data is average of triplicates±SD

FIG. 2B is a schematic representation of the results of HPLCchromatographic analyses of SP304 samples after incubation with (A) heatinactivated SIF for 300 min or with (B) SIF for 120 min. The incubationwith SIF completely converted SP-304 into another peptide eluting at 9.4min, as indicated by *. Arrows indicate the position of SP-304.

FIG. 3 is a schematic representation of the possible degradationproducts of SP-304.

FIG. 4 shows stimulation of cGMP synthesis in T84 cells by the truncatedpeptides of SP-304. Thus, SP-338 has the same peptide sequence as SP-304except that it lacks Leu at the C-terminus. Similarly, SP-327, SP-329and SP-331 have Leu at their C-termini deleted relative to theircorresponding parents, SP-326, SP-328 and SP-330. Peptides wereevaluated for their abilities to stimulate cGMP synthesis in T84 cells.The results are expressed as an average of duplicates.

FIG. 5 shows stimulation of cGMP synthesis in T84 cells by SP-304 andsimilar peptides. Cells were exposed to peptide analogs for 30 min andcell lysates were used to determine intracellular cGMP levels. Resultsare expressed as an average of triplicates±SD.

FIG. 6 shows stimulation of cGMP synthesis in T84 cells by SP-339 andother peptides. T84 Cells were exposed to the indicated peptide for 30min and cell lysates were used to determine intracellular cGMP levels.Results are expressed as an average of triplicates±SD.

FIG. 7A shows stability of SP-333 against digestion with simulatedintestinal fluid (SIF) for indicated times. The control sample marked asC120 was produced by incubating peptides with heat inactivated SIF.Samples from the incubations were removed and heated at 95° C. for 5 minto inactivate digestive enzymes and then used to stimulate cyclic GMPsynthesis in T84 cells. The cGMP stimulation activity at 0 min was takenas 100% in each set. The data is average of triplicates±SD.

FIG. 7B shows stability of SP-332 against digestion with simulatedintestinal fluid (SIF) for indicated times. The control sample marked asC120 was produced by incubating peptides with heat inactivated SIF.Samples from the digestions were removed and heated at 95° C. for 5 minto inactivate digestive enzymes and then used to stimulate cyclic GMPsynthesis in T84 cells. The cGMP stimulation activity at 0 min was takenas 100% in each set. The data is average of triplicates±SD.

FIG. 7C shows stability of SP-304 against digestion with simulatedintestinal fluid (SIF) for indicated times. The control samples markedas C0 and C60 were produced by incubating peptides with heat inactivatedSIF. Samples from the digestions were removed and heated at 95° C. for 5min to inactivate digestive enzymes and then used to stimulate cyclicGMP synthesis in T84 cells. The cGMP stimulation activity at 0 min wastaken as 100% in each set. The data is average of 3 determinations±SD.

FIG. 7D shows HPLC analysis of samples of SP-304 at 0 (FIG. 7D-1) and 60minutes (FIG. 7D-2) following incubation with SIF. Arrow indicates theelution position of SP-304 peptide. The data clearly shows that theSP-304 peak eluting at 14.3 min completely vanished and two new peaksemerged at 7.4 and 10.3 minutes. These new peptide peaks represent thepossible degradation products of SP-304.

FIG. 7E shows HPLC analysis of samples of SP-332 at 0 (FIG. 7E-1) and120 minutes (FIG. 7E-2) following incubation with SIF. Arrow indicatesthe elution position of SP-332 peptide. The data shows that the peptideSP-332 eluting at 14.8 minutes was not changed following incubation withSIF, suggesting that SP-332 is not sensitive to proteolysis by proteasespresent in SIF.

FIG. 7F shows HPLC analysis of samples of SP-333 at 0 (FIG. 7F-1) and120 minutes (FIG. 7F-2) following incubation with SIF. Arrows indicatethe elution position of SP-333. The data show that peptide SP-333,eluting at 14.8 minutes, was not changed following incubation with SIF,suggesting that SP-333 is not sensitive to proteolysis by proteasespresent in SIF during the 120 minute incubation period.

FIG. 8 shows stimulation of cGMP synthesis in T84 cells by thepeggylated analogs of SP-333. T84 cells were exposed to the indicatedpeptides for 30 min and cell lysates were used to determineintracellular cGMP levels. Results are expressed as an average oftriplicates±SD.

FIG. 9 shows stimulation of cGMP synthesis in T84 cells by SP-304 (0.1μM) either alone or in combination with the phosphodiesterase (PDE)inhibitors Sulindac Sulfone (100 μM) or Zaprinast (100 μM). T84 cellswere exposed to various treatments, as indicated, for 30 min and thecell lysates were used to determine the intracellular cGMP levels.Results are expressed as an average of duplicates.

FIG. 10 shows stimulation of cGMP synthesis in T84 cells by SP-304 (0.1or 1.0 μM) either alone or in combination with incrementalconcentrations of phosphodiesterase (PDE) inhibitors, as indicated(upper panel: sulindac sulfone; lower panel: zaprinast). T84 cells wereexposed to various treatments, as indicated, for 30 min and the celllysates were used to determine the intracellular cGMP levels. Resultsare expressed as an average of duplicates.

FIG. 11 shows stimulation of cGMP synthesis in T84 by SP-333 (0.1 or 1.0μM) either alone or in combination with incremental concentrationsZaprinast, as indicated. T84 cells were exposed to various treatments,as indicated, for 30 min and the cell lysates were used to determine theintracellular cGMP levels. Results are expressed as an average ofduplicates.

FIG. 12 shows stimulation of cGMP synthesis in T84 by SP-333 (0.1 μM)either alone or in combination with incremental concentrations SulindacSulfone, as indicated. T84 cells were exposed to various treatments, asindicated, for 30 min and the cell lysates were used to determine theintracellular cGMP levels. Results are expressed as an average ofduplicates.

FIG. 13 shows a schematic of the maintenance of intracellularconcentrations of cGMP levels. The intracellular levels of cGMP can bemaintained by stimulating its synthesis via the activation of GC-C andby inhibiting its degradation by cGMP-PDE. Thus, a combination of a GC-Cagonist with an inhibitor of PDE may produce a synergistic effect toenhance levels of cGMP in tissues and organs.

DETAILED DESCRIPTION

The present invention is based upon the development of agonists ofguanylate cyclase-C (GC-C). The agonists are analogs of uroguanylin andbacterial ST peptides and have superior properties such as for examplehigh resistance to degradation at the N-terminus and C-terminus fromcarboxypeptidases and/or by other proteolytic enzymes such as thosepresent in the stimulated human intestinal juices and human gastricjuices.

The GC-C is expressed on various cells including on gastrointestinalepithelial cells, and on extra-intestinal tissues including kidney,lung, pancreas, pituitary, adrenal, developing liver, heart and male andfemale reproductive tissues (reviewed in Vaandrager 2002 Mol CellBiochem 230:73-83). The GC-C is a key regulator of fluid and electrolytebalance in the intestine and kidney. In the intestine, when stimulated,the GC-C causes an increase in intestinal epithelial cGMP. This increasein cGMP causes a decrease in water and sodium absorption and an increasein chloride and potassium ion secretion, leading to changes inintestinal fluid and electrolyte transport and increased intestinalmotility.

The gualylate cyclase-C agonists according to the invention include SEQID NO:2-54, and SEQ ID NO: 57-98 and are summarized below in Table I andTable II. The gualylate cyclase-C agonists according to the inventionare collectively referred to herein as “GCRA peptides”.

TABLE I  GCRA peptides Position  of Disulfide SEQ Name bonds StructureID NO SP-304 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Va⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶1 C7:C15 SP-326 C3:C11,Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵2 C6:C14 SP-327 C2:C10,Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴3 C5:C13 SP-328 C2:C10,Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴4 C5:C13 SP-329 C2:C10,Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 5C5:C13 SP-330 C1:C9,Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³ 6C4:C12 SP-331 C1:C9,Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-A1a⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹² 7 C4:C12SP332 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶8 C7:C15 SP--333 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-9 C7:C15 dLeu¹⁶ SP-334 C4:C12,dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-10 C7:C15 dLeu¹⁶ SP-335 C4:C12,dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-11 C7:C15 dLeu¹⁶ SP-336 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶12 C7:C15 SP-337 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-13 C7:C15 dLeu¹⁶ SP-338 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹⁻Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵14 C7:C15 SP-342 C4:C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys7-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-15 C7:C15 dLeu¹⁶-PEG3 SP-343 C4:C12,PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-16 C7:C15 dLeu¹⁶-PEG3 SP-344 C4:C12,PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-17 C7:C15 Cys¹⁵-dLeu¹⁶-PEG3 SP-347 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-18 C7:C15 dLeu¹⁶-PEG3 SP-348 C4:C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-19 C7:C15 dLeu¹⁶ SP-350 C4:C12,PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-20 C7:C15 dLeu¹⁶ SP-352 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-21 C7:C15 dLeu¹⁶-PEG3 SP-358 C4:C12,PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-22 C7:C15 Cys¹⁵-dLeu¹⁶-PEG3 SP-359 C4:C12,PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-23 C7:C15 Cys¹⁵-dLeu¹⁶ SP-360 C4:C12,dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-24 C7:C15 dLeu¹⁶-PEG3 SP-361 C4:C12,dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-25 C7:C15 dLeu¹⁶-PEG3 SP-362 C4:C12,PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-26 C7:C15 Cys¹⁵-dLeu¹⁶ SP-368 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-27 C7:C15 dNal¹⁶ SP-369 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-28 C7:C15 dLeu¹⁶ SP-370 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-29 C7:C15 Orn¹⁵-dLeu¹⁶ SP-371 C4:C12,dAsn¹-Asp²-Glu³⁻Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶30 C7:C15 SP-372 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-31 C7:C15 dLeu¹⁶ N1 C4:C12,PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-32 C7:C15 dLeu¹⁶-PEG3 N2 C4:C12,PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-33 C7:C15 dLeu¹⁶ N3 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-34 C7:C15 dLeu¹⁶-PEG3 N4 C4:C12,PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-35 C7:C15 Cys¹⁵-dLeu¹⁶-PEG3 N5 C4:C12,PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-36 C7:C15 Cys¹⁵-dLeu¹⁶ N6 C4:C12,dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-37 C7:C15 dLeu¹⁶-PEG3 N7 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Th^(r13)-Gly¹⁴-Cys¹⁵-Ser¹⁶38 C7:C15 N8 C4:C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹1-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-39 C7:C15 Ser¹⁶-PEG3 N9 C4:C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-40 C7:C15 Ser¹⁶ N10 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-41 C7:C15 PEG3 N11 C4:C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-42 C7:C15 dSer¹⁶-PEG3 N12 C4:C12,PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-43 C7:C15 dSer¹⁶ N13 C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-44 C7:C15 dSer¹⁶-PEG3 Formula C4:C12,Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶45 I C7:C15 Formula C4:C12,Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa10-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶46 II C7:C15 Formula 4:12,Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵-Xaan247 III 7:15 Formula 4:12,Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaan248 IV 7:15 Formula C4:C12,Asn¹-Asp2-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶49 V) C7:C15 Formula C4:C12,dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-50 VI C7:C15 Xaa¹⁶ Formula C4:C12,dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-51 VII C7:C15 Xaa¹⁶ Formula C4:C12,dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-52 VII C7:C15 Xaa¹⁶ (NEW) Formula C4:C12,dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-53 VIII C7:C15 Xaa¹⁶ (NEW) Formula C4:C12,dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-54 IX C7:C15 Xaa¹⁶

TABLE II  GCRA Peptides Position of SEQ Disulfide ID Name bondsStructure NO: SP-339 C1:C6,Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 C2:C10, C5:13 SP-340 C1:C6,Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56C2:C10, C5:13 SP-349 C1:C6,PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-57 C2:C10, Tyr¹⁴-PEG3 C5:13 SP-353 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-58 C4:C12, Cys¹⁵-Tyr¹⁶ C7:15 SP-354 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-59 C4:C12, Cys¹⁵-Tyr¹⁶ C7:15 SP-355 C1:C6,Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 C2:C10, C5:13 SP-357 C1:C6,PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-61 C2:C10, Tyr¹⁴ C5:13 SP-374 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-62 C4:C12, Cys¹⁵-Tyr¹⁶ C7:15 SP-375 C3:C8,Asn¹-Phe²-Cys³-Cys4-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-63 C4:C12, Cys¹⁵-dTyr¹⁶ C7:15 SP-376 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-64 C4:C12, Cys¹⁵-Tyr¹⁶ C7:15 SP-377 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-65 C4:C12, Cys¹⁵-dTyr¹⁶ C7:15 SP-378 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-66 C4:C12, Cys¹⁵-dTyr¹⁶ C7:15 SP-379 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-67 C4:C12, Cys¹⁵-Tyr¹⁶ C7:15 SP-380 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-68 C4:C12, Cys¹⁵-dTyr¹⁶ C7:15 SP-381 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-69 C4:C12, Cys¹⁵-dTyr¹⁶ C7:15 SP-382 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-70 C4:C12, Cys¹⁵-Tyr¹⁶ C7:15 SP-383 C3:C8,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-71 C4:C12, Cys¹⁵-dTyr¹⁶ C7:15 SP384 C1:C6,Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-72 C2:C10, PEG3 C5:13 N14 C1:C6,PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-73 C2:C10, PEG3 C5:13 N15 C1:C6,PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 C2:C10, C5:13 N16 C1:C6,Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 C2:C10, C5:13 N17 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-76 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 C7:15 N18 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-77 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7:15 N19 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹⁻Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-78 C4:C12, Cys¹⁵-Tyr¹⁶-PEG3 C7:15 N20 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-79 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 C7:15 N21 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-80 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7:15 N22 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-81 C4:C12, Cys¹⁵-Tyr¹⁶-PEG3 C7:15 N23 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-82 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 C7:15 N24 C3:C8,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-83 C4:C12, Gly¹⁴-Cys¹⁵-Tyr¹⁶ C7:15 N25 C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-84 C4:C12, Cys¹⁵-Tyr¹⁶-PEG3 C7:15 N26 C1:C6,Cys¹-Cys²-Glu3-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 C2:C10, C5:13 N27 C1:C6,Cys¹-Cys²-Glu3-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴86 C2:C10, C5:13 N28 C1:C6,Cys¹-Cys²-Glu3-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87C2:C10, C5:13 N29 C1:C6,Cys¹-Cys²-Glu3-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88C2:C10, C5:13 N30 1:6, 2:10,Pen¹-Pen²-Glu3-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³-Tyr¹⁴89 5:13 N31 1:6, 2:10,Pen¹-Pen²-Glu3-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 905:13 Formula X C9:C14,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹²-Cys¹³-Cys¹⁴-91 C10:C18, Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Aaa²² C13:21Formula XI C9:C14,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-92 C10:C18, Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Aaa²² C13:21Formula C3:C8,Asn¹-Phe²-Cys³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Cys⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-93 XII C4:C12, Cys¹⁵-Xaa¹⁶ C7:15 Formula 3:8, 4:12,Asn¹-Phe²-Pen³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Pen⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys12-Xaa¹³-Xaa¹⁴-94 XIII C:15 Cys¹⁵-Xaa¹⁶ Formula 3:8, 4:12,Asn¹-Phe²-Maa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Maa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³- 95XIV 7:15 Xaa¹⁴-Maa¹⁵-Aaa¹⁶ Formula 1:6, 2:10,Maa¹-Maa²-Glu3-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 XV 5:13 Formula 1:6, 2:10,Maa¹-Maa²-Glu3-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³- 97XVI 5:13 Formula 1:6, 2:10,Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-98 XVII 5:13 Xaa_(n2)

The GCRA peptides described herein bind the guanylate cyclase C (GC-C)and stimulate intracellular production of cyclic guanosine monophosphate(cGMP). Optionally, the GCRA peptides induce apoptosis. In some aspects,the GCRA peptides stimulate intracellular cGMP production at higherlevels than naturally occurring GC-C agonists (e.g., uroguanylin,guanylin, and ST peptides) and/or SP-304. For example, the GCRA peptidesof the invention stimulate 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared to naturally occurring GC-C agonists and/orSP-304. The terms induced and stimulated are used interchangeablythroughout the specification. The GCRA peptides described herein aremore stable than naturally occurring GC-C agonists and/or SP-304. Bymore stable it is meant that the peptide degrade less and/or more slowlyin simulated gastrointestinal fluid and/or simulated intestinal fluidcompared to naturally occurring GC-C agonists and/or SP-304. Forexample, the GCRA peptide of the invention degrade 2%, 3%, 5%, 10%, 15%,20%, 30%, 40%, 50%, 75%, 90% or less compared to naturally occurringGC-C agonists and/or SP-304.

The GCRA peptides described herein have therapeutic value in thetreatment of a wide variety of disorders and conditions including forexample gastrointestinal disorders, inflammatory disorders, lungdisorders, cancer, cardiac disorders, eye disorders, oral disorders,blood disorders, liver disorders, skin disorders, prostate disorders,endocrine disorders, increasing gastrointestinal motility and obesity.Gastointestinal disorders include for example, irritable bowel syndrome(IBS), non-ulcer dyspepsia, chronic intestinal pseudo-obstruction,functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux,gastroesophageal reflux disease (GERD)ileus (e.g., post-operativeileus), gastroparesis, heartburn (high acidity in the GI tract),constipation (e.g., constipation associated with use of medications suchas opioids, osteoarthritis drugs, osteoporosis drugs; post surgicalconstipation, constipation associated with neuropathic disorders.Inflammatory disorders include tissue and organ inflammation such askidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); pancreaticinflammation (e.g., pancreatis), lung inflammation (e.g., bronchitis orasthma) or skin inflammation (e.g., psoriasis, eczema). Lung Disordersinclude for example chronic obstructive pulmonary disease (COPD), andfibrosis. Cancer includes tissue and organ carcinogenesis includingmetatases such as for example gastrointestinal cancer, (e.g., gastriccancer, esophageal cancer, pancreatic cancer colorectal cancer,intestinal cancer, anal cancer, liver cancer, gallbladder cancer, orcolon cancer; lung cancer; thyroid cancer; skin cancer (e.g., melanoma);oral cancer; urinary tract cancer (e.g. bladder cancer or kidneycancer); blood cancer (e.g. myeloma or leukemia) or prostate cancer.Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high tryglycerides. Liverdisorders include for example cirrhosis and fibrosis. Eye disordersinclude for example increased intra-ocular pressure, glaucoma, dry eyesretinal degeneration, disorders of tear glands or eye inflammation. Skindisorders include for example xerosis. Oral disorders include forexample dry mouth (xerostomia), Sjögren's syndrome, gum diseases (e.g.,periodontal disease), or salivary gland duct blockage or malfunction.Prostate disorders include for example Benign prostatic hyperplasia(BPH). Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

As used herein, the term “guanylate cyclase C (GC-C)” refers to theclass of guanylate cyclase C receptor on any cell type to which theinventive agonist peptides or natural agonists described herein bind. Asused herein, “intestinal guanylate cyclase receptor” is foundexclusively on epithelial cells lining the GI mucosa. Uroguanylin,guanylin, and ST peptides are expected to bind to these receptors andmay induce apoptosis. The possibility that there may be differentreceptors for each agonist peptide is not excluded. Hence, the termrefers to the class of guanylate cyclase receptors on epithelial cellslining the GI mucosa.

As used herein, the term “GCR agonist” is meant to refer to peptidesand/or other compounds that bind to an intestinal guanylate cyclase Cand stimulate fluid and electrolyte transport. This term also coversfragments and pro-peptides that bind to GC-C and stimulate fluid andwater secretion.

As used herein, the term “substantially equivalent” is meant to refer toa peptide that has an amino acid sequence equivalent to that of thebinding domain where certain residues may be deleted or replaced withother amino acids without impairing the peptide's ability to bind to anintestinal guanylate cyclase receptor and stimulate fluid andelectrolyte transport.

Addition of carriers (e.g., phosphate-buffered saline or PBS) and othercomponents to the composition of the present invention is well withinthe level of skill in this art. In addition to the compound, suchcompositions may contain pharmaceutically acceptable carriers and otheringredients known to facilitate administration and/or enhance uptake.Other formulations, such as microspheres, nanoparticles, liposomes, andimmunologically-based systems may also be used in accordance with thepresent invention. Other examples include formulations with polymers(e.g., 20% w/v polyethylene glycol) or cellulose, or entericformulations.

The present invention is based upon several concepts. The first is thatthere is a cGMP-dependent mechanism which regulates the balance betweencellular proliferation and apoptosis and that a reduction in cGMPlevels, due to a deficiency of uroguanylin/guanylin and/or due to theactivation of cGMP-specific phosphodiesterases, is an early and criticalstep in neoplastic transformation. A second concept is that the releaseof arachidonic acid from membrane phospholipids, which leads to theactivation of cytoplasmic phospholipase A2 (cPLA2), cyclooxygenase-2(COX-2) and possibly 5-lipoxygenase (5-LO) during the process ofinflammation, is down-regulated by a cGMP-dependent mechanism, leadingto reduced levels of prostaglandins and leukotrienes, and thatincreasing intracellular levels of cGMP may therefore produce ananti-inflammatory response. In addition, a cGMP-dependent mechanism, isthought to be involved in the control of proinflammatory processes.Therefore, elevating intracellular levels of cGMP may be used as a meansof treating and controlling gastrointestinal disorders, inflammatorydisorders, lung disorders, cancer, cardiac disorders, eye disorders,oral disorders, blood disorders, liver disorders, skin disorders,prostate disorders, endocrine disorders, increasing gastrointestinalmotility and obesity. Gastointestinal disorders include for example,irritable bowel syndrome (IBS), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastroesophageal reflux disease (GERD)ileus(e.g., post-operative ileus), gastroparesis, heartburn (high acidity inthe GI tract), constipation (e.g., constipation associated with use ofmedications such as opioids, osteoarthritis drugs, osteoporosis drugs;post surgical constipation, constipation associated with neuropathicdisorders. Inflammatory disorders include tissue and organ inflammationsuch as kidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); pancreaticinflammation (e.g., pancreatis), lung inflammation (e.g., bronchitis orasthma) or skin inflammation (e.g., psoriasis, eczema). Lung Disordersinclude for example COPD and fibrosis. Cancer includes tissue and organcarcinogenesis including metatases such as for example gastrointestinalcancer, (e.g., gastric cancer, esophageal cancer, pancreatic cancercolorectal cancer, intestinal cancer, anal cancer, liver cancer,gallbladder cancer, or colon cancer; lung cancer; thyroid cancer; skincancer (e.g., melanoma); oral cancer; urinary tract cancer (e.g. bladdercancer or kidney cancer); blood cancer (e.g. myeloma or leukemia) orprostate cancer. Cardiac disorders include for example, congestive heartfailure, trachea cardia hypertension, high cholesterol, or hightryglycerides. Liver disorders include for example cirrhosis andfibrosis. Eye disorders include for example increased intra-ocularpressure, glaucoma, dry eyes retinal degeneration, disorders of tearglands or eye inflammation. Skin disorders include for example xerosis.Oral disorders include for example dry mouth (xerostomia), Sjögren'ssyndrome, gum diseases (e.g., periodontal disease), or salivary glandduct blockage or malfunction. Prostate disorders include for exampleBenign prostatic hyperplasia (BPH). Endocrine disorders include forexample diabetes mellitus, hyperthyroidism, hypothyroidism, and cysticfibrosis.

Without intending to be bound by any theory, it is envisioned that iontransport across the plasma membrane may prove to be an importantregulator of the balance between cell proliferation and apoptosis thatwill be affected by agents altering cGMP concentrations. Uroguanylin hasbeen shown to stimulate K+ efflux, Ca++ influx and water transport inthe gastrointestinal tract (3). Moreover, atrial natriuretic peptide(ANP), a peptide that also binds to a specific guanylate cyclasereceptor, has also been shown to induce apoptosis in rat mesangialcells, and to induce apoptosis in cardiac myocytes by a cGMP mechanism(21-24).

Binding of the present agonists to a guanylate cyclase receptorstimulates production of cGMP. This ligand-receptor interaction, viaactivation of a cascade of cGMP-dependent protein kinases and CFTR,induces apoptosis in target cells. Therefore, administration of thenovel peptides defined by SEQ ID NO:2-54, and SEQ ID NO: 57-98, as shownin Tables I and II, or peptides similar to uroguanylin, or guanylin orE. coli ST peptide are useful in eliminating or, at least retarding, theonset of gastrointestinal disorders, inflammatory disorders, lungdisorders, cancer, cardiac disorders, eye disorders, oral disorders,blood disorders, liver disorders, skin disorders, prostate disorders,endocrine disorders, increasing gastrointestinal motility and obesity.Gastointestinal disorders include for example, irritable bowel syndrome(IBS), non-ulcer dyspepsia, chronic intestinal pseudo-obstruction,functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux,gastroesophageal reflux disease (GERD), ileus inflammation (e.g.,post-operative ileus), gastroparesis, heartburn (high acidity in the GItract), constipation (e.g., constipation associated with use ofmedications such as opioids, osteoarthritis drugs, osteoporosis drugs;post surgical constipation, constipation associated with neuropathicdisorders. Inflammatory disorders include tissue and organ inflammationsuch as kidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); pancreaticinflammation (e.g., pancreatis), lung inflammation (e.g., bronchitis orasthma) or skin inflammation (e.g., psoriasis, eczema). Lung Disordersinclude for example chronic obstructive pulmonary disease (COPD), andfibrosis. Cancer includes tissue and organ carcinogenesis includingmetatases such as for example gastrointestinal cancer, (e.g., gastriccancer, esophageal cancer, pancreatic cancer colorectal cancer,intestinal cancer, anal cancer, liver cancer, gallbladder cancer, orcolon cancer; lung cancer; thyroid cancer; skin cancer (e.g., melanoma);oral cancer; urinary tract cancer (e.g. bladder cancer or kidneycancer); blood cancer (e.g. myeloma or leukemia) or prostate cancer.Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high tryglycerides. Liverdisorders include for example cirrhosis and fibrosis. Eye disordersinclude for example increased intra-ocular pressure, glaucoma, dry eyesretinal degeneration, disorders of tear glands or eye inflammation. Skindisorders include for example xerosis. Oral disorders include forexample dry mouth (xerostomia), Sjögren's syndrome, gum diseases (e.g.,periodontal disease), or salivary gland duct blockage or malfunction.Prostate disorders include for example Benign prostatic hyperplasia(BPH). Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

Uroguanylin is a circulating peptide hormone with natriuretic activityand has been found to stimulate fluid and electrolyte transport in amanner similar to another family of heat stable enterotoxins (STpeptides) secreted by pathogenic strains of E. coli and other entericbacteria that activate guanylate cyclase receptor and cause secretorydiarrhea. Unlike bacterial ST peptides, the binding of uroguanylin toguanylate cyclase receptor is dependent on the physiological pH of thegut. Therefore, uroguanylin is expected to regulate fluid andelectrolyte transport in a pH dependent manner and without causingsevere diarrhea.

GCRA Peptides

In one aspect, the invention provides a GCRA peptide. The GCRA peptidesare analogues uroguanylin and bacterial ST peptide. No particular lengthis implied by the term “peptide”. In some embodiments, the GCRA peptideis less than 25 amino acids in length, e.g., less than or equal to 20,15, 14, 13, 12, 11, 10, or 5 amino acid in length.

The GCRA peptides can be polymers of L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into an D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence. For example a GCRA peptide includes the sequence of SEQ ID NO:SEQ ID NO:2-54, and SEQ ID NO: 57-98. In various embodiments, the GCRApeptide includes the amino acid sequence of SEQ ID NO:45-54 and SEQ IDNO:87-98 where the peptide induces cGMP production by a cell. In variousembodiments the GCRA peptide of the invention includes the amino acidsequence according to Formulas I-IX (e.g. SEQ ID NO:45-54) with theproviso that the GCRA peptide is not SEQ ID NO:1. In further embodimentsthe GCRA peptide of the invention include the amino acid sequenceaccording to Formulas X-XVII (e.g. SEQ ID NO:87-98) with the provisothat the GCRA peptide is not SEQ ID NO:55 or SEQ ID NO:56. By inducingcGMP production is meant that the GCRA peptide induces the production ofintracellular cGMP. Intracellular cGMP is measured by methods known inthe art. For example, the GCRA peptide of the invention stimulate 5%,10%, 20%, 30%, 40%, 50%, 75%, 90% or more intracellular cGMP compared tonaturally occurring GC-C agonists. Optionally, the GCRA peptides of theinvention of the invention stimulate 5%, 10%, 20%, 30%, 40%, 50%, 75%,90% or more intracellular cGMP compared SP-304 (SEQ ID NO:1). In furtherembodiments, the GCRA peptide stimulates apoptosis, e.g., programmedcell death or activate the cystic fibrosis transmembrane conductanceregulator (CFTR). In some embodiments the GCRA peptides described hereinare more stable than naturally occurring GC-C agonists and/or SP-304(SEQ ID NO:1), SP-339 (SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56). By morestable it is meant that the peptide degrade less and/or more slowly insimulated gastric fluid and/or simulated intestinal fluid compared tonaturally occurring GC-C agonists and/or SP-304. For example, the GCRApeptide of the invention degrade 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%,50%, 75%, 90% or less compared to naturally occurring GC-C agonistsand/or SP-304, SP-339 or SP-340.

As used herein PEG3, 3 PEG, is meant to denote polyethylene glycol suchas include aminoethyloxy-ethyloxy-acetic acid (AeeA). As used herein,(e.g., in Formulas I-XVII, SEQ ID NO:45-54 and SEQ ID NO:87-98) X_(aa)is any any natural, unnatural amino acid or amino acid analogue; M_(aa)is a Cysteine (Cys), Penicillamine (Pen) homocysteine, or3-mercaptoproline; Xaa_(n1) is meant to denote an amino acid sequence ofany any natural, unnatural amino acid or amino acid analogue that isone, two or three residues in length; Xaa_(n2) is meant to denote anamino acid sequence of any any natural, unnatural amino acid or aminoacid analogue that is zero or one residue in length; and Xaa_(n3) ismeant to denote an amino acid sequence of any any natural, unnaturalamino acid or amino acid analogue that is zero, one, two, three, four,five or six residues in length. Additionally, any amino acid representedby Xaa, Xaa_(n1), Xaa_(n2, or) Xaa_(n3) may be an L-amino acid, aD-amino acid, a methylated amino acid or any combination of thereof.Optionally, any GCRA peptide represented by Formulas I-VII may containon or more polyethylene glycol residues at the N-terminus, C-terminus orboth. An exemplary polyethylene glycol includeaminoethyloxy-ethyloxy-acetic acid and polymers thereof.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula I, wherein at at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or the aminoacid at position 16 is a serine. Preferably, the amino acid at position16 of Formula I is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 of Formula I is a d-leucine or ad-serine. Optionally, one or more of the amino acids at position 1-3 ofFormula I are D-amino acids or methylated amino acids or a combinationof D-amino acids or methylated amino acids. For example, Asn1, Asp2 orGlu3 (or a combination thereof) of Formula I is a D-amino acids or amethylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula I is a leucine, serine or tyrosine.

In alternative embodiments, GCRA peptides include peptides containingcontaining the amino acid sequence of Formula II, wherein at least oneamino acid of Formula II is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted by Xaa_(n2) of Formula II is aD-amino acid or a methylated amino acid. In some embodiments the aminoacid denoted by Xaa_(n2) of Formula II is a leucine, d-leucine, serineor d-serine. Preferably, the one or more of the amino acids denoted byXaa_(n1) of Formula II is a D-amino acid or a methylated amino acid.Preferably, the amino acid at position Xaa⁶ of Formula II is a leucine,serine or tyrosine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula III, wherein 1) at at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or 2) Maa isnot a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, d-leucine, serine or d-serine. Preferably, the one or more ofthe amino acids denoted by Xaa_(n1) of Formula III is a D-amino acid ora methylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, serine or tyrosine.

In other embodiments, GCRA peptides include peptides containingcontaining the amino acid sequence of Formula IV, wherein at least oneamino acid of Formula IV is a D-amino acid or a methylated amino acidand/or 2) Maa is not a cysteine. Preferably, the Xaa_(n2) of Formula IVis a D-amino acid or a methylated amino acid. In some embodiments theamino acid denoted by Xaa_(n2) of Formula IV is a leucine, d-leucine,serine or d-serine. Preferably, the one or more of the amino acidsdenoted by Xaa_(n1) of Formula IV is a D-amino acid or a methylatedamino acid. Preferably, the amino acid denoted Xaa⁶ of Formula IV is aleucine, serine or tyrosine. In further embodiments, GCRA peptidesinclude peptides containing containing the amino acid sequence ofFormula V, wherein at at least one amino acid of Formula V is a D-aminoacid or a methylated amino acid. Preferably, the amino acid at position16 of Formula V is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 (i.e., Xaa¹⁶) of Formula V is ad-leucine or a d-serine. Optionally, one or more of the amino acids atposition 1-3 of Formula V are D-amino acids or methylated amino acids ora combination of D-amino acids or methylated amino acids. For example,Asn1, Asp2 or Glu3 (or a combination thereof) of Formula V is a D-aminoacids or a methylated amino acid. Preferably, the amino acid denoted atXaa⁶ of Formula V is a leucine, serine or tyrosine.

In additional embodiments, GCRA peptides include peptides containingcontaining the amino acid sequence of Formula VI, VII, VIII, IX.Preferably, the amino acid at position 6 of Formula VI, VII, VIII, IX.is a leucine, serine or tyrosine. In some aspects the amino acid atposition 16 of Formula VI, VII, VIII, IX is a leucine or a serine.Preferably, the amino acid at position 16 of Formula V is a D-amino acidor a methylated amino acid.

In preferred embodiments, the GCRA peptide is SP-332 (SEQ ID NO:8),SP-333 (SEQ ID NO:9) or SP-334 (SEQ ID NO:10).

In additional embodiments, GCRA peptides include peptides containingcontaining the amino acid sequence of Formula X, XI, XII, XIII, XIV, XV,XVI or XVII. Optionally, one or more amino acids of Formulas X, XI, XII,XIII, XIV, XV, XVI or XVII is a D-amino acid or a methylated amino acid.Preferably, the amino acid at the carboxy terminus of the peptidesaccording to Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is aD-amino acid or a methylated amino acid. For example the amino acid atthe carboxy terminus of the peptides according to Formulas X, XI, XII,XIII, XIV, XV, XVI or XVII is a D-tyrosine

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenyalanine or a serine. Most preferably the amino acid denoted by Xaa⁶of Formula XIV is a phenyalanine or a serine. Preferably, the amino aciddenoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, phenyalanineor a serine. Most preferably, the amino acid position Xaa⁴ of Formula V,XVI or XVII is a phenyalanine or a serine.

In preferred embodiments, the GCRA peptide is SP-353 (SEQ ID NO:58) orSP-354 (SEQ ID NO:59).

In certain embodiments, one or more amino acids of the GCRA peptides canbe replaced by a non-naturally occurring amino acid or a naturally ornon-naturally occurring amino acid analog. There are many amino acidsbeyond the standard 20 (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI). Some arenaturally-occurring others are not. (See, for example, Hunt, TheNon-Protein Amino Acids: In Chemistry and Biochemistry of the AminoAcids, Barrett, Chapman and Hall, 1985). For example, an aromatic aminoacid can be replaced by 3,4-dihydroxy-L-phenylalanine,3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) ornor-tyrosine (norTyr). Phg and norTyr and other amino acids includingPhe and Tyr can be substituted by, e.g., a halogen, —CH3, —OH, —CH2NH3,—C(O)H, —CH2CH3, —CN, —CH2CH2CH3, —SH, or another group. Any amino acidcan be substituted by the D-form of the amino acid.

With regard to non-naturally occurring amino acids or naturally andnon-naturally occurring amino acid analogs, a number of substitutions inthe polypeptide and agonists described herein are possible alone or incombination.

For example, glutamine residues can be substituted withgamma-Hydroxy-Glu or gamma-Carboxy-Glu. Tyrosine residues can besubstituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp. Proline residuescan be substituted with homopro (L-pipecolic acid); hydroxy-Pro;3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1, 2, 3 Alanine residues can be substituted with alpha-substituted orN-methylated amino acid such as alpha-amino isobutyric acid (aib),L/D-alpha-ethylalanine (L/D-isovaline), L/D-methylvaline, orL/D-alpha-methylleucine or a non-natural amino acid such asbeta-fluoro-Ala. Alanine can also be substituted with: n=0, 1, 2, 3Glycine residues can be substituted with alpha-amino isobutyric acid(aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Further examples of unnatural amino acids include: an unnatural analogof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α,α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc β-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, 0-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid(Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AIB);cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; acetamidomethyl protected amino acids andpegylated amino acids. Further examples of unnatural amino acids andamino acid analogs can be found in U.S. 20030108885, U.S. 20030082575,US20060019347 (paragraphs 410-418) and the references cited therein. Thepolypeptides of the invention can include further modificationsincluding those described in US20060019347, paragraph 589. ExemplaryGCRA peptides which include a n0n-naturally occurring amino acid includefor example SP-368 and SP-369.

In some embodiments, an amino acid can be replaced by anaturally-occurring, non-essential amino acid, e.g., taurine.

Alternatively, the GCRA peptides are cyclic peptides. GCRA cyclicpeptide are prepared by methods known in the art. For example,macrocyclization is often accomplished by forming an amide bond betweenthe peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39:51-124 (1988). In various aspects the GCRA peptides are [4,12; 7,15]bicycles.

In some GCRA peptides one or both members of one or both pairs of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al. 1996Int J Pept Protein Res 48:274); β, β dimethylcysteine (Hunt et al. 1993Int JPept Protein Res 42:249) or diaminopropionic acid (Smith et al.1978 J Med Chem 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In addition, one or more disulfide bonds can be replaced by alternativecovalent cross-links, e.g., an amide linkage (—CH2CH(O)NHCH 2- or—CH2NHCH(O)CH 2-), an ester linkage, a thioester linkage, a lactambridge, a carbamoyl linkage, a urea linkage, a thiourea linkage, aphosphonate ester linkage, an alkyl linkage (—CH2CH2CH2CH2-), an alkenyllinkage(—CH 2CH═CHCH 2-), an ether linkage (—CH2CH2OCH2- or—CH2OCH2CH2-), a thioether linkage (—CH2CH2SCH2- or —CH2SCH2CH2-), anamine linkage (—CH2CH2NHCH2- or —CH2NHCH 2CH2-) or a thioamide linkage(—CH2CH(S)HNHCH 2- or —CH2NHCH(S)CH 2-). For example, Ledu et al. (ProcNat'l Acad. Sci. 100:11263-78, 2003) describe methods for preparinglactam and amide cross-links. Exemplary GCRA peptides which include alactam bridge include for example SP-370.

The GCRA peptides can have one or more conventional polypeptide bondsreplaced by an alternative bond. Such replacements can increase thestability of the polypeptide. For example, replacement of thepolypeptide bond between a residue amino terminal to an aromatic residue(e.g. Tyr, Phe, Trp) with an alternative bond can reduce cleavage bycarboxy peptidases and may increase half-life in the digestive tract.Bonds that can replace polypeptide bonds include: a retro-inverso bond(C(O)—NH instead of NH—C(O); a reduced amide bond (NH—CH2); athiomethylene bond (S—CH2 or CH2-S); an oxomethylene bond (O—CH 2 orCH2-O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)—NH); atrans-olefine bond (CH═CH); a fluoro substituted trans-olefine bond(CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O) wherein R is H orCH3; and a fluoro-ketomethylene bond (C(O)—CFR or CFR—C(O) wherein R isH or F or CH3.

The GCRA peptides can be modified using standard modifications.Modifications may occur at the amino (N-), carboxy (C-) terminus,internally or a combination of any of the preceeding. In one aspectdescribed herein, there may be more than one type of modification on thepolypeptide. Modifications include but are not limited to: acetylation,amidation, biotinylation, cinnamoylation, farnesylation, formylation,myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr),stearoylation, succinylation, sulfurylation and cyclisation (viadisulfide bridges or amide cyclisation), and modification by Cys3 orCys5. The GCRA peptides described herein may also be modified by2,4-dinitrophenyl (DNP), DNP-lysine, modification by7-Amino-4-methyl-coumarin (AMC), flourescein, NBD(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B, EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl,dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine). The GCRApeptides described herein may also be conjugated to, for example,polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight orbranched alkyl groups); fatty acid radicals; combinations of PEG, alkylgroups and fatty acid radicals (See, U.S. Pat. No. 6,309,633; Soltero etal., 2001 Innovations in Pharmaceutical Technology 106-110); BSA and KLH(Keyhole Limpet Hemocyanin). The addition of PEG and other polymerswhich can be used to modify polypeptides of the invention is describedin US20060 19347 section IX.

Also included in the invention are peptides that biologically orfunctional equivalent to the peptides described herein. The term“biologically equivalent” or functional equivalent” is intended to meanthat the compositions of the present invention are capable ofdemonstrating some or all of the cGMP production modulatory effects.

GCRA peptides can also include derivatives of GCRA peptides which areintended to include hybrid and modified forms of GCRA peptides in whichcertain amino acids have been deleted or replaced and modifications suchas where one or more amino acids have been changed to a modified aminoacid or unusual amino acid and modifications such as glycosylation solong the modified form retains the biological activity of GCRA peptides.By retaining the biological activity, it is meant that cGMP and orapoptosis is induced by the GCRA peptide, although not necessarily atthe same level of potency as that of a naturally-occurring GCRA peptideidentified.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a GCRApolypeptide is replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a GCRA coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedto identify mutants that retain activity.

Also included within the meaning of substantially homologous is any GCRApeptide which may be isolated by virtue of cross-reactivity withantibodies to the GCRA peptide.

Preparation of GCRA Peptides

GCRA peptides are easily prepared using modern cloning techniques, ormay be synthesized by solid state methods or by site-directedmutagenesis. A GCRA peptide may include dominant negative forms of apolypeptide.

Chemical synthesis may generally be performed using standard solutionphase or solid phase peptide synthesis techniques, in which a peptidelinkage occurs through the direct condensation of the amino group of oneamino acid with the carboxy group of the other amino acid with theelimination of a water molecule. Peptide bond synthesis by directcondensation, as formulated above, requires suppression of the reactivecharacter of the amino group of the first and of the carboxyl group ofthe second amino acid. The masking substituents must permit their readyremoval, without inducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor. Solidphase peptide synthesis uses an insoluble polymer for support duringorganic synthesis. The polymer-supported peptide chain permits the useof simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc., 1963, 85:2149, which involves assembling a linearpeptide chain on a resin support using protected amino acids. Solidphase peptide synthesis typically utilizes either the Boc or Fmocstrategy, which are well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the synthesis.In addition, solid phase synthesis is generally most suitable whenpeptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Alternatively the GCRA peptides are produced by modern cloningtechniques For example, the GCRA peptides are produced either inbacteria including, without limitation, E. coli, or in other existingsystems for polypeptide or protein production (e.g., Bacillus subtilis,baculovirus expression systems using Drosophila Sf9 cells, yeast orfilamentous fungal expression systems, mammalian cell expressionsystems), or they can be chemically synthesized. If the GCRA peptide orvariant peptide is to be produced in bacteria, e.g., E. coli, thenucleic acid molecule encoding the polypeptide may also encode a leadersequence that permits the secretion of the mature polypeptide from thecell. Thus, the sequence encoding the polypeptide can include the presequence and the pro sequence of, for example, a naturally-occurringbacterial ST polypeptide. The secreted, mature polypeptide can bepurified from the culture medium.

The sequence encoding a GCRA peptide described herein can be insertedinto a vector capable of delivering and maintaining the nucleic acidmolecule in a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof. Construction of a vector containing a nucleicacid described herein can be followed by transformation of a host cellsuch as a bacterium. Suitable bacterial hosts include but are notlimited to, E. coli, B subtilis, Pseudomonas, Salmonella. The geneticconstruct also includes, in addition to the encoding nucleic acidmolecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCRA peptide describedherein can also be fused to a nucleic acid encoding a polypeptideaffinity tag, e.g., glutathione S-transferase (GST), maltose E bindingprotein, protein A, FLAG tag, hexa-histidine, myc tag or the influenzaHA tag, in order to facilitate purification. The affinity tag orreporter fusion joins the reading frame of the polypeptide of interestto the reading frame of the gene encoding the affinity tag such that atranslational fusion is generated. Expression of the fusion gene resultsin translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCRA peptides and variants described herein inprotein expression systems other than bacteria, and well known to thoseskilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

Therapeutic Methods

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated that is mediated by guanylate cyclasereceptor agonists. Disorders mediated by the guanylate cyclase receptoragonists include gastrointestinal disorders, inflammatory disorders,lung disorders, cancer, cardiac disorders, eye disorders, oraldisorders, blood disorders, liver disorders, skin disorders, prostatedisorders, endocrine disorders, increasing gastrointestinal motility andobesity. Gastointestinal disorders include for example, irritable bowelsyndrome (IBS), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastroesophageal reflux disease (GERD)ileus(e.g., post-operative ileus), gastroparesis, heartburn (high acidity inthe GI tract), constipation (e.g., constipation associated with use ofmedications such as opioids, osteoarthritis drugs, osteoporosis drugs;post surgical constipation, constipation associated with neuropathicdisorders. Inflammatory disorders include tissue and organ inflammationsuch as kidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); pancreaticinflammation (e.g., pancreatis), lung inflammation (e.g., bronchitis orasthma) or skin inflammation (e.g., psoriasis, eczema). Lung Disordersinclude for example chronic obstructive pulmonary disease (COPD), andfibrosis. Cancer includes tissue and organ carcinogenesis includingmetatases such as for example gastrointestinal cancer, (e.g., gastriccancer, esophageal cancer, pancreatic cancer colorectal cancer,intestinal cancer, anal cancer, liver cancer, gallbladder cancer, orcolon cancer; lung cancer; thyroid cancer; skin cancer (e.g., melanoma);oral cancer; urinary tract cancer (e.g. bladder cancer or kidneycancer); blood cancer (e.g. myeloma or leukemia) or prostate cancer.Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high tryglycerides. Liverdisorders include for example cirrhosis and fibrosis. Eye disordersinclude for example increased intra-ocular pressure, glaucoma, dry eyesretinal degeneration, disorders of tear glands or eye inflammation. Skindisorders include for example xerosis. Oral disorders include forexample dry mouth (xerostomia), Sjögren's syndrome, gum diseases (e.g.,periodontal disease), or salivary gland duct blockage or malfunction.Prostate disorders include for example benign prostatic hyperplasia(BPH). Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

The term “treatment” refers to reducing or alleviating symptoms in asubject, preventing symptoms from worsening or progressing, and/orpreventing disease in a subject who is free therefrom. For a givensubject, improvement in a symptom, its worsening, regression, orprogression may be determined by any objective or subjective measure.Efficacy of the treatment may be measured as an improvement in morbidityor mortality (e.g., lengthening of survival curve for a selectedpopulation). Thus, effective treatment would include therapy of existingdisease, control of disease by slowing or stopping its progression,prevention of disease occurrence, reduction in the number or severity ofsymptoms, or a combination thereof. The effect may be shown in acontrolled study using one or more statistically significant criteria.

Intracellular cGMP induced by exposing, e.g., contacting a tissue (e.g.,gastrointestinals tissue) or cell with GCRA agonists. GC-C receptors areexpressed throughout the GI tract starting from esophagus, duodenum,jejunum, ilium, caecum and colon. Human colon cancer cell lines (T81,CaCo-2 and HT-29) also express GC-C receptors. By inducing is meant anincrease in cGMP production compared to a tissue or cell that has notbeen in contact with GCRA peptide or variant. Tissues or cells aredirectly contacted with a GCRA peptide or variant. Alternatively, theGCRA peptide or variant is administered systemically. GCRA peptide orvariant are administered in an amount sufficient to increaseintracellular cGMP concentration. cGMP production is measured by acell-based assay known in the art (25).

Disorders are treated, prevented or alleviated by administering to asubject, e.g., a mammal such as a human in need thereof, atherapeutically effective dose of a GCRA peptide. The GCRA peptides maybe in a pharmaceutical composition in unit dose form, together with oneor more pharmaceutically acceptable excipients. The term “unit doseform” refers to a single drug delivery entity, e.g., a tablet, capsule,solution or inhalation formulation. The amount of peptide present shouldbe sufficient to have a positive therapeutic effect when administered toa patient (typically, between 10 μg and 3 g). What constitutes a“positive therapeutic effect” will depend upon the particular conditionbeing treated and will include any significant improvement in acondition readily recognized by one of skill in the art.

The GCRA peptides can be administered alone or in combination with otheragents. For example the GCRA peptides can be administered in combinationwith inhibitors of cGMP dependent phosphodiesterase, such as, forexample, suldinac sulfone, zaprinast, motapizone, vardenafil orsildenifil; one or more other chemotherapeutic agents; oranti-inflammatory drugs such as, for example, steroids or non-steroidalanti-inflammatory drugs (NSAIDS), such as aspirin.

Combination therapy can be achieved by administering two or more agents,e.g., a GCRA peptide described herein and another compound, each ofwhich is formulated and administered separately, or by administering twoor more agents in a single formulation. Other combinations are alsoencompassed by combination therapy. For example, two agents can beformulated together and administered in conjunction with a separateformulation containing a third agent. While the two or more agents inthe combination therapy can be administered simultaneously, they neednot be. For example, administration of a first agent (or combination ofagents) can precede administration of a second agent (or combination ofagents) by minutes, hours, days, or weeks. Thus, the two or more agentscan be administered within minutes of each other or within 1, 2, 3, 6,9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or10 weeks of each other. In some cases even longer intervals arepossible. While in many cases it is desirable that the two or moreagents used in a combination therapy be present in within the patient'sbody at the same time, this need not be so.

The GCRA peptides described herein may be combined withphosphodiesterase inhibitors, e.g., sulindae sulfone, Zaprinast,sildenafil, vardenafil or tadalafil to further enhance levels of cGMP inthe target tissues or organs.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

Combination therapy can also include the administration of two or moreagents via different routes or locations. For example, (a) one agent isadministered orally and another agents is administered intravenously or(b) one agent is administered orally and another is administeredlocally. In each case, the agents can either simultaneously orsequentially. Approximated dosages for some of the combination therapyagents described herein are found in the “BNF Recommended Dose” columnof tables on pages 11-17 of WO01/76632 (the data in the tables beingattributed to the March 2000 British National Formulary) and can also befound in other standard formularies and other drug prescribingdirectories. For some drugs, the customary prescribed dose for anindication will vary somewhat from country to country.

The GCRA peptides, alone or in combination, can be combined with anypharmaceutically acceptable carrier or medium. Thus, they can becombined with materials that do not produce an adverse, allergic orotherwise unwanted reaction when administered to a patient. The carriersor mediums used can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients (which include starches, polyols, granulating agents,microcrystalline cellulose (e.g. celphere, Celphere Beads®), diluents,lubricants, binders, disintegrating agents, and the like), etc. Ifdesired, tablet dosages of the disclosed compositions may be coated bystandard aqueous or nonaqueous techniques.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a GCRA agonist) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods ofpreparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. Such as mannitol, fructooligosaccharides, polyethylene glycoland other excepients. They can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Compositions of the present invention may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, glidants,anti-adherents, anti-static agents, surfactants (wetting agents),anti-oxidants, film-coating agents, and the like. Any such optionalingredient must be compatible with the compound described herein toinsure the stability of the formulation.

The composition may contain other additives as needed, including forexample lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, raffnose, maltitol, melezitose, stachyose, lactitol,palatinite, starch, xylitol, mannitol, myoinositol, and the like, andhydrates thereof, and amino acids, for example alanine, glycine andbetaine, and polypeptides and proteins, for example albumen.

Examples of excipients for use as the pharmaceutically acceptablecarriers and the pharmaceutically acceptable inert carriers and theaforementioned additional ingredients include, but are not limited tobinders, fillers, disintegrants, lubricants, anti-microbial agents, andcoating agents such as: BINDERS: corn starch, potato starch, otherstarches, gelatin, natural and synthetic gums such as acacia, xanthan,sodium alginate, alginic acid, other alginates, powdered tragacanth,guar gum, cellulose and its derivatives (e.g., ethyl cellulose,cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose), polyvinyl pyrrolidone (e.g., povidone, crospovidone,copovidone, etc), methyl cellulose, Methocel, pre-gelatinized starch(e.g., STARCH 1500® and STARCH 1500 LM®, sold by Colorcon, Ltd.),hydroxypropyl methyl cellulose, microcrystalline cellulose (FMCCorporation, Marcus Hook, Pa., USA), or mixtures thereof, FILLERS: talc,calcium carbonate (e.g., granules or powder), dibasic calcium phosphate,tribasic calcium phosphate, calcium sulfate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,dextrose, fructose, honey, lactose anhydrate, lactose monohydrate,lactose and aspartame, lactose and cellulose, lactose andmicrocrystalline cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose &amp; guar gum, molasses, sucrose, ormixtures thereof, DISINTEGRANTS: agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, clays, otheralgins, other celluloses, gums (like gellan), low-substitutedhydroxypropyl cellulose, or mixtures thereof, LUBRICANTS: calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, sodium stearyl fumarate, vegetable based fattyacids lubricant, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil andsoybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloidsilica gel (AEROSIL 200, W.R. Grace Co., Baltimore, Md. USA), acoagulated aerosol of synthetic silica (Deaussa Co., Piano, Tex. USA), apyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass. USA), ormixtures thereof, ANTI-CAKING AGENTS: calcium silicate, magnesiumsilicate, silicon dioxide, colloidal silicon dioxide, talc, or mixturesthereof, ANTIMICROBIAL AGENTS: benzalkonium chloride, benzethoniumchloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridiniumchloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben,methylparaben, phenol, phenylethyl alcohol, phenoxyethanol,phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate,propylparaben, sodium benzoate, sodium dehydroacetate, sodiumpropionate, sorbic acid, thimersol, thymo, or mixtures thereof, andCOATING AGENTS: sodium carboxymethyl cellulose, cellulose acetatephthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropyl methylcellulose (hypromellose), hydroxypropylmethyl cellulose phthalate, methylcellulose, polyethylene glycol,polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,carnauba wax, microcrystalline wax, gellan gum, maltodextrin,methacrylates, microcrystalline cellulose and carrageenan or mixturesthereof.

The formulation can also include other excipients and categories thereofincluding but not limited to L-histidine, Pluronic®, Poloxamers (such asLutrol® and Poloxamer 188), ascorbic acid, glutathione, permeabilityenhancers (e.g. lipids, sodium cholate, acylcarnitine, salicylates,mixed bile salts, fatty acid micelles, chelators, fatty acid,surfactants, medium chain glycerides), protease inhibitors (e.g. soybeantrypsin inhibitor, organic acids), pH lowering agents and absorptionenhancers effective to promote bioavailability (including but notlimited to those described in U.S. Pat. No. 6,086,918 and U.S. Pat. No.5,912,014), creams and lotions (like maltodextrin and carrageenans);materials for chewable tablets (like dextrose, fructose, lactosemonohydrate, lactose and aspartame, lactose and cellulose, maltodextrin,maltose, mannitol, microcrystalline cellulose and guar gum, sorbitolcrystalline); parenterals (like mannitol and povidone); plasticizers(like dibutyl sebacate, plasticizers for coatings, polyvinylacetatephthalate); powder lubricants (like glyceryl behenate); soft gelatincapsules (like sorbitol special solution); spheres for coating (likesugar spheres); spheronization agents (like glyceryl behenate andmicrocrystalline cellulose); suspending/gelling agents (likecarrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone,sodium starch glycolate, xanthan gum); sweeteners (like aspartame,aspartame and lactose, dextrose, fructose, honey, maltodextrin, maltose,mannitol, molasses, sorbitol crystalline, sorbitol special solution,sucrose); wet granulation agents (like calcium carbonate, lactoseanhydrous, lactose monohydrate, maltodextrin, mannitol, microcrystallinecellulose, povidone, starch), caramel, carboxymethylcellulose sodium,cherry cream flavor and cherry flavor, citric acid anhydrous, citricacid, confectioner's sugar, D&C Red No. 33, D&C Yellow #10 AluminumLake, disodium edetate, ethyl alcohol 15%, FD&C Yellow No. 6 aluminumlake, FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2aluminum lake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No. 10, glycerolpalmitostearate, glyceryl monostearate, indigo carmine, lecithin,manitol, methyl and propyl parabens, mono ammonium glycyrrhizinate,natural and artificial orange flavor, pharmaceutical glaze, poloxamer188, Polydextrose, polysorbate 20, polysorbate 80, polyvidone,pregelatinized corn starch, pregelatinized starch, red iron oxide,saccharin sodium, sodium carboxymethyl ether, sodium chloride, sodiumcitrate, sodium phosphate, strawberry flavor, synthetic black ironoxide, synthetic red iron oxide, titanium dioxide, and white wax.

Solid oral dosage forms may optionally be treated with coating systems(e.g. Opadry® fx film coating system, for example Opadry® blue(OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white (YS-1-7040), andblack ink (S-1-8 106).

The agents either in their free form or as a salt can be combined with apolymer such as polylactic-glycoloic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat.No. 4,767,628), poly(ε-caprolactone) and poly(alkylene oxide) (U.S.20030068384) to create a sustained release formulation. Suchformulations can be used to implants that release a polypeptide oranother agent over a period of a few days, a few weeks or several monthsdepending on the polymer, the particle size of the polymer, and the sizeof the implant (See, e.g., U.S. Pat. No. 6,620,422). Other sustainedrelease formulations and polymers for use in are described in EP 0 467389 A2, WO 93/24150, U.S. Pat. No. 5,612,052, WO 97/40085, WO 03/075887,WO 01/01964A2, U.S. Pat. No. 5,922,356, WO 94/155587, WO 02/074247A2, WO98/25642, U.S. Pat. No. 5,968,895, U.S. Pat. No. 6,180,608, U.S.20030171296. U.S. 20020176841, U.S. Pat. No. 5,672,659, U.S. Pat. No.5,893,985, U.S. Pat. No. 5,134,122, U.S. 5,192,741, U.S. Pat. No.5,192,741, U.S. Pat. No. 4,668,506, U.S. Pat. No. 4,713,244, U.S. Pat.No. 5,445,832 U.S. Pat. No. 4,931,279, U.S. 5,980,945, WO 02/058672, WO9726015, WO 97/04744, and US200200 19446. In such sustained releaseformulations microparticles (Delie and Blanco-Prieto 2005 Molecule10:65-80) of polypeptide are combined with microparticles of polymer.One or more sustained release implants can be placed in the largeintestine, the small intestine or both. U.S. Pat. No. 6,011,01 and WO94/06452 describe a sustained release formulation providing eitherpolyethylene glycols (i.e. PEG 300 and PEG 400) or triacetin. WO03/053401 describes a formulation which may both enhance bioavailabilityand provide controlled release of the agent within the GI tract.Additional controlled release formulations are described in WO 02/38129,EP 326151, U.S. Pat. No. 5,236,704, WO 02/30398, WO 98/13029; U.S.20030064105, U.S. 20030138488A1, U.S. 20030216307A1, U.S. Pat. No.6,667,060, WO 01/49249, WO 01/49311, WO 01/49249, WO 01/49311, and U.S.Pat. No. 5,877,224 materials which may include those described inWO04041195 (including the seal and enteric coating described therein)and pH-sensitive coatings that achieve delivery in the colon includingthose described in U.S. Pat. No. 4,910,021 and WO9001329. U.S. Pat. No.4,910,021 describes using a pH-sensitive material to coat a capsule.WO9001329 describes using pH-sensitive coatings on beads containingacid, where the acid in the bead core prolongs dissolution of thepH-sensitive coating. U.S. Pat. No. 5,175,003 discloses a dual mechanismpolymer mixture composed of pH-sensitive enteric materials andfilm-forming plasticizers capable of conferring permeability to theenteric material, for use in drug-delivery systems; a matrix pelletcomposed of a dual mechanism polymer mixture permeated with a drug andsometimes covering a pharmaceutically neutral nucleus; a membrane-coatedpellet comprising a matrix pellet coated with a dual mechanism polymermixture envelope of the same or different composition; and apharmaceutical dosage form containing matrix pellets. The matrix pelletreleases acid-soluble drugs by diffusion in acid pH and bydisintegration at pH levels of nominally about 5.0 or higher. The GCRApeptideds described herein may be formulated in the pH triggeredtargeted control release systems described in WO04052339. The agentsdescribed herein may be formulated according to the methodologydescribed in any of WO03105812 (extruded hyrdratable polymers);WO0243767 (enzyme cleavable membrane translocators); WO03007913 andWO03086297 (mucoadhesive systems); WO02072075 (bilayer laminatedformulation comprising pH lowering agent and absorption enhancer);WO04064769 (amidated polypeptides); WO05063156 (solid lipid suspensionwith pseudotropic and/or thixotropic properties upon melting);WO03035029 and WO03035041 (erodible, gastric retentive dosage forms);U.S. Pat. No. 5,007,790 and U.S. Pat. No. 5,972,389 (sustained releasedosage forms); WO041 1271 1 (oral extended release compositions);WO05027878, WO02072033, and WO02072034 (delayed release compositionswith natural or synthetic gum); WO05030182 (controlled releaseformulations with an ascending rate of release); WO05048998(microencapsulation system); U.S. Pat. No. 5,952,314 (biopolymer); U.S.Pat. No. 5,108,758 (glassy amylose matrix delivery); U.S. Pat. No.5,840,860 (modified starch based delivery). JP10324642 (delivery systemcomprising chitosan and gastric resistant material such as wheat gliadinor zein); U.S. Pat. No. 5,866,619 and U.S. Pat. No. 6,368,629(saccharide containing polymer); U.S. Pat. No. 6,531,152 (describes adrug delivery system containing a water soluble core (Ca pectinate orother water-insoluble polymers) and outer coat which bursts (e.g.hydrophobic polymer-Eudragrit)); U.S. Pat. No. 6,234,464; U.S. Pat. No.6,403,130 (coating with polymer containing casein and high methoxypectin; WO0174 175 (Maillard reaction product); WO05063206 (solubilityincreasing formulation); WO040 19872 (transferring fusion proteins).

The GCRA peptides described herein may be formulated usinggastrointestinal retention system technology (GIRES; MerrionPharmaceuticals). GIRES comprises a controlled-release dosage forminside an inflatable pouch, which is placed in a drug capsule for oraladministration. Upon dissolution of the capsule, a gas-generating systeminflates the pouch in the stomach where it is retained for 16-24 hours,all the time releasing agents described herein.

The GCRA peptides described herein can be formulated in an osmoticdevice including the ones disclosed in U.S. Pat. No. 4,503,030, U.S.Pat. No. 5,609,590 and U.S. Pat. No. 5,358,502. U.S. Pat. No. 4,503,030discloses an osmotic device for dispensing a drug to certain pH regionsof the gastrointestinal tract. More particularly, the invention relatesto an osmotic device comprising a wall formed of a semi-permeable pHsensitive composition that surrounds a compartment containing a drug,with a passageway through the wall connecting the exterior of the devicewith the compartment. The device delivers the drug at a controlled ratein the region of the gastrointestinal tract having a pH of less than3.5, and the device self-destructs and releases all its drug in theregion of the gastrointestinal tract having a pH greater than 3.5,thereby providing total availability for drug absorption. U.S. Pat. Nos.5,609,590 and 5,358,502 disclose an osmotic bursting device fordispensing a beneficial agent to an aqueous environment. The devicecomprises a beneficial agent and osmagent surrounded at least in part bya semi-permeable membrane. The beneficial agent may also function as theosmagent. The semi-permeable membrane is permeable to water andsubstantially impermeable to the beneficial agent and osmagent. Atrigger means is attached to the semi-permeable membrane (e.g., joinstwo capsule halves). The trigger means is activated by a pH of from 3 to9 and triggers the eventual, but sudden, delivery of the beneficialagent. These devices enable the pH-triggered release of the beneficialagent core as a bolus by osmotic bursting.

Exemplary Agents for Combination Therapy

Analgesic Agents

The GCRA peptides described herein can be used in combination therapywith an analgesic agent, e.g., an analgesic compound or an analgesicpolypeptide. These polypeptides and compounds can be administered withthe GCRA peptides described herein (simultaneously or sequentially).They can also be optionally covalently linked or attached to an agentdescribed herein to create therapeutic conjugates. Among the usefulanalgesic agents are: Ca channel blockers, 5HT receptor antagonists (forexample 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptoragonists (loperamide, fedotozine, and fentanyl), NK1 receptorantagonists, CCK receptor agonists (e.g., loxiglumide), NK1 receptorantagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptakeinhibitors (NSR1), vanilloid and cannabanoid receptor agonists, andsialorphin. Analgesics agents in the various classes are described inthe literature.

Among the useful analgesic polypeptides are sialorphin-relatedpolypeptides, including those comprising the amino acid sequence QHNPR(SEQ ID NO: 99), including: VQHNPR (SEQ ID NO: 100); VRQHNPR (SEQ ID NO:101); VRGQHNPR (SEQ ID NO: 102); VRGPQHNPR (SEQ ID NO: 103); VRGPRQHNPR(SEQ ID NO: 104); VRGPRRQHNPR (SEQ ID NO: 105); and RQHNPR (SEQ ID NO:106). Sialorphin-related polypeptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or polypeptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the polypeptidesdescribed herein in a co-therapy or linked to the polypeptides describedherein, e.g., by a covalent bond. Sialophin and related polypeptides aredescribed in U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO02/051435 A2.

Opioid receptor antagonists and agonists can be administered with theGCRA peptides described herein in co-therapy or linked to the agentdescribed herein, e.g., by a covalent bond. For example, opioid receptorantagonists such as naloxone, naltrexone, methyl nalozone, nalmefene,cypridime, beta funaltrexamine, naloxonazine, naltrindole, andnor-binaltorphimine are thought to be useful in the treatment of IBS. Itcan be useful to formulate opioid antagonists of this type is a delayedand sustained release formulation such that initial release of theantagonist is in the mid to distal small intestine and/or ascendingcolon. Such antagonists are described in WO 01/32180 A2. Enkephalinpentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine) (SEQ ID NO: 122)is an agonist of the mu and delta opioid receptors and is thought to beuseful for increasing intestinal motility {Eur. J. Pharm. 219:445,1992), and this polypeptide can be used in conjunction with thepolypeptides described herein. Also useful is trimebutine which isthought to bind to mu/delta/kappa opioid receptors and activate releaseof motilin and modulate the release of gastrin, vasoactive intestinalpolypeptide, gastrin and glucagons. Kappa opioid receptor agonists suchas fedotozine, asimadoline, and ketocyclazocine, and compounds describedin WO03/097051 and WO05/007626 can be used with or linked to thepolypeptides described herein. In addition, mu opioid receptor agonistssuch as morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH2; WO 01/019849 A1) (SEQ ID NO: 123) andloperamide can be used.

Tyr-Arg (kyotorphin) is a dipeptide that acts by stimulating the releaseof met-enkephalins to elicit an analgesic effect (J. Biol. Chem.262:8165, 1987). Kyotorphin can be used with or linked to the GCRApeptides described herein.

Chromogranin-derived polypeptide (CgA 47-66; See, e.g., Ghia et al. 2004Regulatory polypeptides 119:199) can be used with or linked to the GCRApeptides described herein.

CCK receptor agonists such as caerulein from amphibians and otherspecies are useful analgesic agents that can be used with or linked tothe GCRA peptides described herein.

Conotoxin polypeptides represent a large class of analgesic polypeptidesthat act at voltage gated calcium channels, NMDA receptors or nicotinicreceptors. These polypeptides can be used with or linked to thepolypeptides described herein. Peptide analogs of thymulin (FRApplication 2830451) can have analgesic activity and can be used with orlinked to the polypeptides described herein.

CCK (CCKa or CCKb) receptor antagonists, including loxiglumide anddexloxiglumide (the R-isomer of loxiglumide) (WO 88/05774) can haveanalgesic activity and can be used with or linked to the polypeptidesdescribed herein.

Other useful analgesic agents include 5-HT4 agonists such as tegaserod(Zelnorm®), mosapride, metoclopramide, zacopride, cisapride, renzapride,benzimidazolone derivatives such as BIMU 1 and BIMU 8, and lirexapride.Such agonists are described in: EP1321 142 A1, WO 03/053432A1, EP 505322A1, EP 505322 B1, U.S. Pat. No. 5,510,353, EP 507672 A1, EP 507672 B1,and U.S. Pat. No. 5,273,983.

Calcium channel blockers such as ziconotide and related compoundsdescribed in, for example, EP625162B1, U.S. Pat. No. 5,364,842, U.S.Pat. No. 5,587,454, U.S. Pat. No. 5,824,645, U.S. Pat. No. 5,859,186,U.S. Pat. No. 5,994,305, U.S. Pat. No. 6,087,091, U.S. Pat. No.6,136,786, WO 93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1,U.S. Pat. No. 5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. No.6,054,429, WO 97/01351 A1, can be used with or linked to thepolypeptides described herein.

Various antagonists of the NK-I, NK-2, and NK-3 receptors (for a reviewsee Giardina et al. 2003. Drugs 6:758) can be can be used with or linkedto the polypeptides described herein.

NK1 receptor antagonists such as: aprepitant (Merck & Co Inc),vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd),SR-48968 (Sanofi Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (GlaxoSmith Kline), TAK-637 (Takeda/Abbot), SR-14033, and related compoundsdescribed in, for example, EP 873753 A1, US 20010006972 A1, US20030109417 A1, WO 01/52844 A1, can be used with or linked to thepolypeptides described herein.

NK-2 receptor antagonists such as nepadutant (Menarini Ricerche SpA),saredutant (Sanoft-Synthelabo), GW597599 (Glaxo Smith Kline), SR-144190(Sanoft-Synthelabo) and UK-290795 (Pfizer Inc) can be used with orlinked to the polypeptides described herein.

NK3 receptor antagonists such as osanetant (SR-142801;Sanoft-Synthelabo), SSR-241586, talnetant and related compoundsdescribed in, for example, WO 02/094187 A2, EP 876347 A1, WO 97/21680A1, U.S. Pat. No. 6,277,862, WO 98/1 1090, WO 95/28418, WO 97/19927, andBoden et al. (J Med. Chem. 39:1664-75, 1996) can be used with or linkedto the polypeptides described herein.

Norepinephrine-serotonin reuptake inhibitors (NSR1) such as milnacipranand related compounds described in WO 03/077897 A1 can be used with orlinked to the polypeptides described herein.

Vanilloid receptor antagonists such as arvanil and related compoundsdescribed in WO 01/64212 A1 can be used with or linked to thepolypeptides described herein.

The analgesic polypeptides and compounds can be administered with thepolypeptides and agonists described herein (simultaneously orsequentially). The analgesic agents can also be covalently linked to thepolypeptides and agonists described herein to create therapeuticconjugates. Where the analgesic is a polypeptide and is covalentlylinked to an agent described herein the resulting polypeptide may alsoinclude at least one trypsin cleavage site. When present within thepolypeptide, the analgesic polypeptide may be preceded by (if it is atthe carboxy terminus) or followed by (if it is at the amino terminus) atrypsin cleavage site that allows release of the analgesic polypeptide.

In addition to sialorphin-related polypeptides, analgesic polypeptidesinclude: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin,lupron, ziconotide, and substance P.

Agents to Treat Gastrointestinal Disorders

Examples of additional therapeutic agents to treat gastrointestinal andother disorders include agents to treat constipation (e.g., a chloridechannel activator such as the bicylic fatty acid, Lubiprostone (formerlyknown as SPI-0211; Sucampo Pharmaceuticals, Inc.; Bethesda, Md.), alaxative (e.g. a bulk-forming laxative (e.g. nonstarch polysaccharides,Colonel Tablet (polycarbophil calcium), Plantago Ovata®, Equalactin®(Calcium Polycarbophil)), fiber (e.g. FIBERCON® (Calcium Polycarbophil),an osmotic laxative, a stimulant laxative (such as diphenylmethanes(e.g. bisacodyl), anthraquinones (e.g. cascara, senna), and surfactantlaxatives (e.g. castor oil, docusates), an emollient/lubricating agent(such as mineral oil, glycerine, and docusates), MiraLax (BraintreeLaboratories, Braintree Mass.), dexloxiglumide (Forest Laboratories,also known as CR 2017 Rottapharm (Rotta Research Laboratorium SpA)),saline laxatives, enemas, suppositories, and CR 3700 (Rottapharm (RottaResearch Laboratorium SpA); acid reducing agents such as proton pumpinhibitors (e.g., omeprazole (Prilosec®), esomeprazole (Nexium®),lansoprazole (Prevacid®), pantoprazole (Protonix®) and rabeprazole(Aciphex®)) and Histamine H2-receptor antagonist (also known as H2receptor blockers including cimetidine, ranitidine, famotidine andnizatidine); prokinetic agents including itopride, octreotide,bethanechol, metoclopramide (Reglan®), domperidone (Motilium®),erythromycin (and derivatives thereof) or cisapride (Propulsid®);Prokineticin polypeptides homologs, variants and chimeras thereofincluding those described in U.S. Pat. No. 7,052,674 which can be usedwith or linked to the polypeptides described herein; pro-motility agentssuch as the vasostatin-derived polypeptide, chromogranin A (4-16) (See,e.g., Ghia et al. 2004 Regulatory polypeptides 121:31) or motilinagonists (e.g., GM-611 or mitemcinal fumarate) or nociceptin/Orphanin FQreceptor modulators (US20050169917); other peptides which can bind toand/or activate GC-C including those described in US20050287067;complete or partial 5HT (e.g. 5HT1, 5HT2, 5HT3, 5HT4) receptor agonistsor antagonists (including 5HT1A antagonists (e.g. AGI-OOl (AGItherapeutics), 5HT2B antagonists (e.g. PGN 1091 and PGNl 164 (PharmageneLaboratories Limited), and 5HT4 receptor agonists (such as tegaserod(ZELNORM®), prucalopride, mosapride, metoclopramide, zacopride,cisapride, renzapride, benzimidazolone derivatives such as BIMU 1 andBIMU 8, and lirexapride). Such agonists/modulators are described in:EP1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, U.S. Pat. No.5,510,353, EP 507672 A1, EP 507672 B1, U.S. Pat. No. 5,273,983, and U.S.Pat. No. 6,951,867); 5HT3 receptor agonists such as MKC-733; and 5HT3receptor antagonists such as DDP-225 (MC1-225; Dynogen Pharmaceuticals,Inc.), cilansetron (Calmactin®), alosetron (Lotronex®), Ondansetron HCl(Zofran®), Dolasetron (ANZEMET®), palonosetron (Aloxi®), Granisetron(Kytril®), YM060 (ramosetron; Astellas Pharma Inc.; ramosetron may begiven as a daily dose of 0.002 to 0.02 mg as described in EP01588707)and ATI-7000 (Aryx Therapeutics, Santa Clara Calif.); muscarinicreceptor agonists; anti-inflammatory agents; antispasmodics includingbut not limited to anticholinergic drugs (like dicyclomine (e.g.Colimex®, Formulex®, Lomine®, Protylol®, Visceral®, Spasmoban®, Bentyl®,Bentylol®), hyoscyamine (e.g. IB-Stat®, Nulev®, Levsin®, Levbid®,Levsinex Timecaps®, Levsin/SL®, Anaspaz®, A-Spas S/L®, Cystospaz®,Cystospaz-M®, Donnamar®, Colidrops Liquid Pediatric®, Gastrosed®, HycoElixir®, Hyosol®, Hyospaz®, Hyosyne®, Losamine®, Medispaz®, Neosol®,Spacol®, Spasdel®, Symax®, Symax SL®), Donnatal (e.g. DonnatalExtentabs®), clidinium (e.g. Quarzan, in combination withLibrium=Librax), methantheline (e.g. Banthine), Mepenzolate (e.g.Cantil), homatropine (e.g. hycodan, Homapin), Propantheline bromide(e.g. Pro-Banthine), Glycopyrrolate (e.g. Robinul®, Robinul Forte®),scopolamine (e.g. Transderm-Scop®, Transderm-V®), hyosine-N-butylbromide(e.g. Buscopan®), Pirenzepine (e.g. Gastrozepin®) Propantheline Bromide(e.g. Propanthel®), dicycloverine (e.g. Merbentyl®), glycopyrroniumbromide (e.g. Glycopyrrolate®), hyoscine hydrobromide, hyoscinemethobromide, methanthelinium, and octatropine); peppermint oil; anddirect smooth muscle relaxants like cimetropium bromide, mebeverine(DUSPATAL®, DUSPATALIN®, COLOFAC MR®, COLOTAL®), otilonium bromide(octilonium), pinaverium (e.g. Dicetel® (pinaverium bromide; Solvay S.A.)), Spasfon® (hydrated phloroglucinol and trimethylphloroglucinol) andtrimebutine (including trimebutine maleate (Modulon®); antidepressants,including but not limited to those listed herein, as well as tricyclicantidepressants like amitriptyline desipramine (Norpramin®), imipramine(Tofranil®), amoxapine (Asendin®), nortriptyline; the selectiveserotonin reuptake inhibitors (SSRTs) like paroxetine (Paxil®),fluoxetine (Prozac®), sertraline (Zoloft®), and citralopram (Celexa®);and others like doxepin (Sinequan®) and trazodone (Desyrel®);centrally-acting analgesic agents such as opioid receptor agonists,opioid receptor antagonists (e.g., naltrexone); agents for the treatmentof Inflammatory bowel disease; agents for the treatment of Crohn'sdisease and/or ulcerative colitis (e.g., alequel (Enzo Biochem, Inc.;Farmingsale, N.Y.), the anti-inflammatory polypeptide RDP58 (Genzyme,Inc.; Cambridge, Mass.), and TRAFICET-EN™ (ChemoCentryx, Inc.; SanCarlos, Calif.); agents that treat gastrointestinal or visceral pain;agents that increase cGMP levels (as described in US20040121994) likeadrenergic receptor antagonists, dopamine receptor agonists and PDE(phosphodiesterase) inhibitors including but not limited to thosedisclosed herein; purgatives that draw fluids to the intestine (e.g.,VISICOL®, a combination of sodium phosphate monobasic monohydrate andsodium phosphate dibasic anhydrate); Corticotropin Releasing Factor(CRF) receptor antagonists (including NBI-34041 (Neurocrine Biosciences,San Diego, Calif.), CRH9-41, astressin, R121919 (Janssen Pharmaceutica),CP154,526, NBI-27914, Antalarmin, DMP696 (Bristol-Myers Squibb)CP-316,311 (Pfizer, Inc.), SB723620 (GSK), GW876008 (Neurocrine/GlaxoSmith Kline), ONO-2333Ms (Ono Pharmaceuticals), TS-041 (Janssen), AAG561(Novartis) and those disclosed in U.S. Pat. No. 5,063,245, U.S. Pat. No.5,861,398, US20040224964, US20040198726, US20040176400, US20040171607,US20040110815, US20040006066, and US20050209253); glucagon-likepolypeptides (glp-1) and analogues thereof (including exendin-4 andGTP-010 (Gastrotech Pharma A)) and inhibitors of DPP-IV (DPP-IV mediatesthe inactivation of glp-1); tofisopam, enantiomerically-pureR-tofisopam, and pharmaceutically-acceptable salts thereof (US20040229867); tricyclic anti-depressants of the dibenzothiazepine typeincluding but not limited to Dextofisopam® (Vela Pharmaceuticals),tianeptine (Stablon®) and other agents described in U.S. Pat. No.6,683,072; (E)-4(1,3bis(cyclohexylmethyl)-1,2,34,-tetrahydro-2,6-diono-9H-purin-8-yl)cinnamicacid nonaethylene glycol methyl ether ester and related compoundsdescribed in WO 02/067942; the probiotic PROBACTRIX® (The BioBalanceCorporation; New York, N.Y.) which contains microorganisms useful in thetreatment of gastrointestinal disorders; antidiarrheal drugs includingbut not limited to loperamide (Imodium, Pepto Diarrhea), diphenoxylatewith atropine (Lomotil, Lomocot), cholestyramine (Questran, Cholybar),atropine (Co-Phenotrope, Diarsed, Diphenoxylate, Lofene, Logen, Lonox,Vi-Atro, atropine sulfate injection) and Xifaxan® (rifaximin; SalixPharmaceuticals Ltd), TZP-201(Tranzyme Pharma Inc.), the neuronalacetylcholine receptor (nAChR) blocker AGI-004 (AGI therapeutics), andbismuth subsalicylate (Pepto-bismol); anxiolytic drugs including but notlimited toAtivan (lorazepam), alprazolam (Xanax®),chlordiazepoxide/clidinium (Librium®, Librax®), clonazepam (Klonopin®),clorazepate (Tranxene®), diazepam (Valium®), estazolam (ProSom®),flurazepam (Dalmane®), oxazepam (Serax®), prazepam (Centrax®), temazepam(Restoril®), triazolam (Halcion®; Bedelix® (Montmorillonite beidellitic;Ipsen Ltd), Solvay SLV332 (ArQuIe Inc), YKP(SK Pharma), Asimadoline(Tioga Pharmaceuticals/Merck), AGI-003 (AGI Therapeutics); neurokininantagonists including those described in US20060040950; potassiumchannel modulators including those described in U.S. Pat. No. 7,002,015;the serotonin modulator AZD7371 (AstraZeneca PIc); M3 muscarinicreceptor antagonists such as darifenacin (Enablex; Novartis AG andzamifenacin (Pfizer); herbal and natural therapies including but notlimited to acidophilus, chamomile tea, evening primrose oil, fennelseeds, wormwood, comfrey, and compounds of Bao-Ji-Wan (magnolol,honokiol, imperatorin, and isoimperatorin) as in U.S. Pat. No.6,923,992; and compositions comprising lysine and an anti-stress agentfor the treatment of irritable bowel syndrome as described in EPO1550443.

Insulin and Insulin Modulating Agents

The GCRA peptides described herein can be used in combination therapywith insulin and related compounds including primate, rodent, or rabbitinsulin including biologically active variants thereof including allelicvariants, more preferably human insulin available in recombinant form.Sources of human insulin include pharmaceutically acceptable and sterileformulations such as those available from Eli Lilly (Indianapolis, Ind.46285) as Humulin™ (human insulin rDNA origin). See, the THE PHYSICIAN'SDESK REFERENCE, 55.sup.th Ed. (2001) Medical Economics, ThomsonHealthcare (disclosing other suitable human insulins).

The GCRA peptides described herein can also be used in combinationtherapy with agents that can boost insulin effects or levels of asubject upon administration, e.g. glipizide and/or rosiglitazone. Thepolypeptides and agonistsdescribed herein can be used in combitherapywith SYMLIN® (pramlintide acetate) and Exenatide® (synthetic exendin-4;a 39 aa polypeptide).

Agents for the Treatment of Postoperative Ileus

The GCRA peptides described herein can also be used in combinationtherapy with agents (e.g., Entereg™ (alvimopan; formerly called adolor/ADL 8-2698), conivaptan and related agents describe in U.S. Pat. No.6,645,959) used for the treatment of postoperative ileus and otherdisorders.

Anti-Hypertensive Agents

The GCRA peptides described herein can be used in combination therapywith an anti-hypertensive agent including but not limited to: (1)diuretics, such as thiazides, including chlorthalidone, chlorthiazide,dichlorophenamide, hydroflumethiazide, indapamide, polythiazide, andhydrochlorothiazide; loop diuretics, such as bumetanide, ethacrynicacid, furosemide, and torsemide; potassium sparing agents, such asamiloride, and triamterene; carbonic anhydrase inhibitors, osmotics(such as glycerin) and aldosterone antagonists, such as spironolactone,epirenone, and the like; (2) beta-adrenergic blockers such asacebutolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol,carteolol, carvedilol, celiprolol, esmolol, indenolol, metaprolol,nadolol, nebivolol, penbutolol, pindolol, propanolol, sotalol,tertatolol, tilisolol, and timolol, and the like; (3) calcium channelblockers such as amlodipine, aranidipine, azelnidipine, barnidipine,benidipine, bepridil, cinaldipine, clevidipine, diltiazem, efonidipine,felodipine, gallopamil, isradipine, lacidipine, lemildipine,lercanidipine, nicardipine, nifedipine, nilvadipine, nimodepine,nisoldipine, nitrendipine, manidipine, pranidipine, and verapamil, andthe like; (4) angiotensin converting enzyme (ACE) inhibitors such asbenazepril; captopril; ceranapril; cilazapril; delapril; enalapril;enalopril; fosinopril; imidapril; lisinopril; losinopril; moexipril;quinapril; quinaprilat; ramipril; perindopril; perindropril; quanipril;spirapril; tenocapril; trandolapril, and zofenopril, and the like; (5)neutral endopeptidase inhibitors such as omapatrilat, cadoxatril andecadotril, fosidotril, sampatrilat, AVE7688, ER4030, and the like; (6)endothelin antagonists such as tezosentan, A308165, and YM62899, and thelike; (7) vasodilators such as hydralazine, clonidine, minoxidil, andnicotinyl alcohol, and the like; (8) angiotensin II receptor antagonistssuch as aprosartan, candesartan, eprosartan, irbesartan, losartan,olmesartan, pratosartan, tasosartan, telmisartan, valsartan, andEXP-3137, FI6828K, and RNH6270, and the like; (9) α/β adrenergicblockers such as nipradilol, arotinolol and amosulalol, and the like;(10) alpha 1 blockers, such as terazosin, urapidil, prazosin,tamsulosin, bunazosin, trimazosin, doxazosin, naftopidil, indoramin, WHP164, and XENOlO, and the like; (11) alpha 2 agonists such as lofexidine,tiamenidine, moxonidine, rilmenidine and guanobenz, and the like; (12)aldosterone inhibitors, and the like; and (13) angiopoietin-2-bindingagents such as those disclosed in WO03/030833. Specificanti-hypertensive agents that can be used in combination withpolypeptides and agonists described herein include, but are not limitedto: diuretics, such as thiazides (e.g., chlorthalidone, cyclothiazide(CAS RN 2259-96-3), chlorothiazide (CAS RN 72956-09-3, which may beprepared as disclosed in U.S. Pat. No. 2,809,194), dichlorophenamide,hydroflumethiazide, indapamide, polythiazide, bendroflumethazide,methyclothazide, polythiazide, trichlormethazide, chlorthalidone,indapamide, metolazone, quinethazone, althiazide (CAS RN 5588-16-9,which may be prepared as disclosed in British Patent No. 902,658),benzthiazide (CAS RN 91-33-8, which may be prepared as disclosed in U.S.Pat. No. 3,108,097), buthiazide (which may be prepared as disclosed inBritish Patent Nos. 861,367), and hydrochlorothiazide), loop diuretics(e.g. bumetanide, ethacrynic acid, furosemide, and torasemide),potassium sparing agents (e.g. amiloride, and triamterene (CAS Number396-01-O)), and aldosterone antagonists (e.g. spironolactone (CAS Number52-01-7), epirenone, and the like); β-adrenergic blockers such asAmiodarone (Cordarone, Pacerone), bunolol hydrochloride (CAS RN31969-05-8, Parke-Davis), acebutolol (±N-[3-Acetyl-4-[2-hydroxy-3-[(1methylethyl)amino]propoxy]phenyl]-butanamide, or(±)-3]-Acetyl-4′-[2-hydroxy-3-(isopropylamino) propoxy]butyranilide),acebutolol hydrochloride (e.g. Sectral®, Wyeth-Ayerst), alprenololhydrochloride (CAS RN 13707-88-5 see Netherlands Patent Application No.6,605,692), atenolol (e.g. Tenormin®, AstraZeneca), carteololhydrochloride (e.g. Cartrol® Filmtab®, Abbott), Celiprolol hydrochloride(CAS RN 57470-78-7, also see in U.S. Pat. No. 4,034,009), cetamololhydrochloride (CAS RN 77590-95-5, see also U.S. Pat. No. 4,059,622),labetalol hydrochloride (e.g. Normodyne®, Schering), esmololhydrochloride (e.g. Brevibloc®, Baxter), levobetaxolol hydrochloride(e.g. Betaxon™ Ophthalmic Suspension, Alcon), levobunolol hydrochloride(e.g. Betagan® Liquifilm® with C CAP® Compliance Cap, Allergan), nadolol(e.g. Nadolol, Mylan), practolol (CAS RN 6673-35-4, see also U.S. Pat.No. 3,408,387), propranolol hydrochloride (CAS RN 318-98-9), sotalolhydrochloride (e.g. Betapace AF™, Berlex), timolol (2-Propanol,l-[(l,l-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-,hemihydrate, (S)-, CAS RN 91524-16-2), timolol maleate(S)-I-[(1,1-dimethylethyl)amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-2-propanol(Z)-2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol(2-Propanol,1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth-ylethyl)amino]-,(±), CAS RN 66722-44-9), bisoprolol fumarate (such as(±)-1-[4-[[2-(1-Methylethoxy)ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol(E)-2-butenedioate (2:1) (salt), e.g., Zebeta™, Lederle Consumer),nebivalol (2H-1-Benzopyran-2-methanol,αα′-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS RN 99200-09-6see also U.S. Pat. No. 4,654,362), cicloprolol hydrochloride, such2-Propanol,1-[4-[2-[(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride(2-Propanol,l-[l-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride(CAS RN 13071-11-9), diacetolol hydrochloride (Acetamide,N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy][phenyl]-,monohydrochloride CAS RN 69796-04-9), dilevalol hydrochloride(Benzamide,2-hydroxy-5-[l-hydroxy-2-[1-methyl-3-phenylpropyl)amino]ethyl]-,monohydrochloride, CAS RN 75659-08-4), exaprolol hydrochloride(2-Propanol,1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]hydrochloride CAS RN59333-90-3), flestolol sulfate (Benzoic acid,2-fluoro-,3-[[2-[aminocarbonyl)amino]-dimethylethyl]amino]-2-hydroxypropylester, (+)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalolhydrochloride (Methanesulfonamide,N-[4-[1-hydroxy-2-(methylamino)propyl]phenyl}-, monohydrochloride CAS RN7701-65-7), metoprolol 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[1-methylethyl)amino]-; CAS RN37350-58-6), metoprolol tartrate (such as 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g.,Lopressor®, Novartis), pamatolol sulfate (Carbamic acid,[2-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenyl]-ethyl]-,methyl ester, (±) sulfate (salt) (2:1), CAS RN 59954-01-7), penbutololsulfate (2-Propanol,1-(2-cyclopentylphenoxy)-3-[1,1-dimethyle-thyl)amino]1, (S)-, sulfate(2:1) (salt), CAS RN 38363-32-5), practolol (Acetamide,N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN6673-35-4;) tiprenolol hydrochloride (Propanol,1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-, hydrochloride,(±), CAS RN 39832-43-4), tolamolol (Benzamide,4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl]amino]ethoxyl]-, CAS RN38103-61-6), bopindolol, indenolol, pindolol, propanolol, tertatolol,and tilisolol, and the like; calcium channel blockers such as besylatesalt of amlodipine (such as3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylatebenzenesulphonate, e.g., Norvasc®, Pfizer), clentiazem maleate(1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-(2S-cis)-,(Z)-2-butenedioate (1:1), see also U.S. Pat. No. 4,567,195), isradipine(3,5-Pyridinedicarboxylic acid,4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethylester,(±)-4(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate,see also U.S. Pat. No. 4,466,972); nimodipine (such as is isopropyl(2-methoxyethyl)1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate,e.g. Nimotop®, Bayer), felodipine (such as ethyl methyl4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-,e.g. Plendil® Extended-Release, AstraZeneca LP), nilvadipine(3,5-Pyridinedicarboxylic acid,2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-,3-methyl5-(1-methylethyl)ester, also see U.S. Pat. No. 3,799,934), nifedipine(such as 3,5-pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, e.g.,Procardia XL® Extended Release Tablets, Pfizer), diltiazem hydrochloride(such as1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis., e.g., Tiazac®, Forest), verapamilhydrochloride (such as benzeneacetronitrile,(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Isoptin® SR, Knoll Labs), teludipine hydrochloride(3,5-Pyridinedicarboxylic acid,2-[(dimethylamino)methyl]4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propenyl]phenyl}-1,4-dihydro-6-methyl-,diethyl ester, monohydrochloride) CAS RN 108700-03-4), belfosdil(Phosphonic acid, [2-(2-phenoxy ethyl)-1,3-propane-diyl]bis-, tetrabutylester CAS RN 103486-79-9), fostedil (Phosphonic acid,[[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester CAS RN 75889-62-2),aranidipine, azelnidipine, barnidipine, benidipine, bepridil,cinaldipine, clevidipine, efonidipine, gallopamil, lacidipine,lemildipine, lercanidipine, monatepil maleate (1-piperazinebutanamide,N-(6,11-dihydrodibenzo(b,e)thiepin-11-yl)₄-(4-fluorophenyl)-, (+)-,(Z)-2-butenedioate (1:1)(±)-N-(6,11-Dihydrodibenzo(b,e)thiep-in-1′-yl)-4-(p-fluorophenyl)-1-piperazinebutyramidemaleate (1:1) CAS RN 132046-06-1), nicardipine, nisoldipine,nitrendipine, manidipine, pranidipine, and the like; T-channel calciumantagonists such as mibefradil; angiotensin converting enzyme (ACE)inhibitors such as benazepril, benazepril hydrochloride (such as3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-aceticacid monohydrochloride, e.g., Lotrel®, Novartis), captopril (such as1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g., Captopril, Mylan,CAS RN 62571-86-2 and others disclosed in U.S. Pat. No. 4,046,889),ceranapril (and others disclosed in U.S. Pat. No. 4,452,790), cetapril(alacepril, Dainippon disclosed in Eur. Therap. Res. 39:671 (1986);40:543 (1986)), cilazapril (Hoffman-LaRoche) disclosed in J. Cardiovasc.Pharmacol. 9:39 (1987), indalapril (delapril hydrochloride(2H-1,2,4-Benzothiadiazine-7-sulfonamide,3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide CAS RN2259-96-3); disclosed in U.S. Pat. No. 4,385,051), enalapril (and othersdisclosed in U.S. Pat. No. 4,374,829), enalopril, enaloprilat,fosinopril, ((such as L-proline,4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy)propoxy](4-phenylbutyl)phosphinyl]acetyl]-, sodium salt, e.g., Monopril,Bristol-Myers Squibb and others disclosed in U.S. Pat. No. 4,168,267),fosinopril sodium (L-Proline,4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-ox-opropoxy)propox), imidapril,indolapril (Schering, disclosed in J. Cardiovasc. Pharmacol. 5:643, 655(1983)), lisinopril (Merck), losinopril, moexipril, moexiprilhydrochloride (3-Isoquinolinecarboxylic acid,2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]aminoF1-oxopropyl]-1,-2,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride,(3S)-CAS RN 82586-52-5), quinapril, quinaprilat, ramipril (Hoechsst)disclosed in EP 79022 and Curr. Ther. Res. 40:74 (1986), perindoprilerbumine (such as2S,3aS,7aS-1-[(S)—N—[(S)-1-Carboxybutyljalanyl]hexahydrô-indolinecarboxylicacid, 1-ethyl ester, compound with tert-butylamine (1:1), e.g., Aceon®,Solvay), perindopril (Servier, disclosed in Eur. J. din. Pharmacol.31:519 (1987)), quanipril (disclosed in U.S. Pat. No. 4,344,949),spirapril (Schering, disclosed in Acta. Pharmacol. Toxicol. 59 (Supp.5): 173 (1986)), tenocapril, trandolapril, zofenopril (and othersdisclosed in U.S. Pat. No. 4,316,906), rentiapril (fentiapril, disclosedin Clin. Exp. Pharmacol. Physiol. 10:131 (1983)), pivopril, YS980,teprotide (Bradykinin potentiator BPP9a CAS RN 35115-60-7), BRL 36,378(Smith Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai, see CA.102:72588v and Jap. J. Pharmacol. 40:373 (1986), CGS 14824 (Ciba-Geigy,3-([1-ethoxycarbonyl-3-phenyl-(1S)-propyl]amino)-2,3,4,5-tetrahydro-2-ox-o-1-(3S)-benzazepine-1-aceticacid HCl, see U.K. Patent No. 2103614), CGS 16,617 (Ciba-Geigy,3(S)-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,-5-tetrahydro-2-oxo-1H-1-benzazepine-1-ethanoicacid, see U.S. Pat. No. 4,473,575), Ru 44570 (Hoechst, seeArzneimittelforschung 34:1254 (1985)), R 31-2201 (Hoffman-LaRoche seeFEBS Lett. 165:201 (1984)), CI925 (Pharmacologist 26:243, 266 (1984)),WY-44221 (Wyeth, see J. Med. Chem. 26:394 (1983)), and those disclosedin US2003006922 (paragraph 28), U.S. Pat. No. 4,337,201, U.S. Pat. No.4,432,971 (phosphonamidates); neutral endopeptidase inhibitors such asomapatrilat (Vanlev®), CGS 30440, cadoxatril and ecadotril, fasidotril(also known as aladotril or alatriopril), sampatrilat, mixanpril, andgemopatrilat, AVE7688, ER4030, and those disclosed in U.S. Pat. No.5,362,727, U.S. Pat. No. 5,366,973, U.S. Pat. No. 5,225,401, U.S. Pat.No. 4,722,810, U.S. Pat. No. 5,223,516, U.S. Pat. No. 4,749,688, U.S.Pat. No. 5,552,397, U.S. Pat. No. 5,504,080, U.S. Pat. No. 5,612,359,U.S. Pat. No. 5,525,723, EP0599444, EP0481522, EP0599444, EP0595610,EP0534363, EP534396, EP534492, EP0629627; endothelin antagonists such astezosentan, A308165, and YM62899, and the like; vasodilators such ashydralazine (apresoline), clonidine (clonidine hydrochloride(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-,monohydrochloride CAS RN 4205-91-8), catapres, minoxidil (loniten),nicotinyl alcohol (roniacol), diltiazem hydrochloride (such as1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis, e.g., Tiazac®, Forest), isosorbide dinitrate(such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g., Isordil®Titradose®, Wyeth-Ayerst), sosorbide mononitrate (such as1,4:3,6-dianhydro-D-glucito-1,5-nitrate, an organic nitrate, e.g.,Ismo®, Wyeth-Ayerst), nitroglycerin (such as 2,3 propanetrioltrinitrate, e.g., Nitrostat® Parke-Davis), verapamil hydrochloride (suchas benzeneacetonitrile, (±)-(alpha)[3-[[2-(3,4 dimethoxyphenyDethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride,e.g., Covera HS® Extended-Release, Searle), chromonar (which may beprepared as disclosed in U.S. Pat. No. 3,282,938), clonitate (Annalen1870 155), droprenilamine (which may be prepared as disclosed inDE2521113), lidoflazine (which may be prepared as disclosed in U.S. Pat.No. 3,267,104); prenylamine (which may be prepared as disclosed in U.S.Pat. No. 3,152,173), propatyl nitrate (which may be prepared asdisclosed in French Patent No. 1,103,113), mioflazine hydrochloride(1-piperazineacetamide,3-(aminocarbonyl)-4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophenyl)-,dihydrochloride CAS RN 83898-67-3), mixidine (Benzeneethanamine,3,4-dimethoxy-N-(1-methyl-2-pyrrolidinylidene)-Pyrrolidine,2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,4-dimethoxyphenethyl)imino]pyrrolidineCAS RN 27737-38-8), molsidomine (1,2,3-Oxadiazolium,5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN25717-80-0), isosorbide mononitrate (D-Glucito-1,1,4:3,6-dianhydro-,5-nitrate CAS RN 16051-77-7), erythrityl tetranitrate(1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-CAS RN 7297-25-8),clonitrate(1,2-Propanediol, 3-chloro-, dinitrate (7CI, 8CI, 9CI) CAS RN2612-33-1), dipyridamole Ethanol,2,2′,2″,2′″-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]tetrakis-CASRN 58-32-2), nicorandil (CAS RN 65141-46-0 3-), pyridinecarboxamide(N-[2-(nitrooxy)ethyl]-Nisoldipine-3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl esterCAS RN 63675-72-9), nifedipine-3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN21829-25-4), perhexyline maleate (Piperidine,2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN 6724-53-4),oxprenolol hydrochloride (2-Propanol,1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-, hydrochlorideCAS RN 6452-73-9), pentrinitrol (1,3-Propanediol,2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6),verapamil (Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-CASRN 52-53-9) and the like; angiotensin II receptor antagonists such as,aprosartan, zolasartan, olmesartan, pratosartan, FI6828K, RNH6270,candesartan (1H-Benzimidazole-7-carboxylic acid,2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]4-yl]methyl]-CAS RN139481-59-7), candesartan cilexetil(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]-1H-benzimidazolecarboxylate, CAS RN 145040-37-5, U.S. Pat. No. 5,703,110 and U.S. Pat.No. 5,196,444), eprosartan(341-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl)propenoic acid, U.S. Pat. No. 5,185,351 and U.S. Pat. No. 5,650,650),irbesartan (2-n-butyl-3-[[2′-(1h-tetrazol-5-yl)biphenyl-4-yl]methyl]1,3-diazazspiro[4,4]non-1-en-4-one,U.S. Pat. No. 5,270,317 and U.S. Pat. No. 5,352,788), losartan(2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole,potassium salt, U.S. Pat. No. 5,138,069, U.S. Pat. No. 5,153,197 andU.S. Pat. No. 5,128,355), tasosartan(5,8-dihydro-2,4-dimethyl-8-[(2′-(1H-tetrazol-5-yl)[l,r-biphenyl]4-yl)methyl]-pyrido[2,3-d]pyrimidin-7(6H)-one,U.S. Pat. No. 5,149,699), telmisartan(4′-[(1,4-dimethyl-2′-propyl-(2,6′-bi-1H-benzimidazol)-r-yl)]-[1,1′-biphenyl]-2-carboxylicacid, CAS RN 144701-48-4, U.S. Pat. No. 5,591,762), milfasartan,abitesartan, valsartan (Diovan® (Novartis),(S)—N-valeryl-N-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine, U.S.Pat. No. 5,399,578), EXP-3137(2-N-butyl-4-chloro-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylicacid, U.S. Pat. No. 5,138,069, U.S. Pat. No. 5,153,197 and U.S. Pat. No.5,128,355),3-(2′-(tetrazol-5-yl)-l,r-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine,4′[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-2-yl]-benzimidazol-1-yl]-methyl]-l,r-biphenyl]-2-carboxylicacid,2-butyl-6-(1-methoxy-1-methylethyl)-2-[2′-)1H-tetrazol-5-yl)biphenyl-4-ylmethyl]quinazolin-4(3H)-one,3-[2′-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5-b]pyridine,2-butyl-4-chloro-1-[(2′-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-carboxylicacid, 2-butyl-4-chloro-1-[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl]methyl]-1H-imidazole-5-carboxylicacid-1-(ethoxycarbonyl-oxy)ethyl ester potassium salt, dipotassium2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,1′-biphenyl)-4-yl]methyl]-1H-imidazole-5-carboxylate,methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophencarboxylate,5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol-5-ylphenyl)]pyridine,6-butyl-2-(2-phenylethyl)-5[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-methyl]pyrimidin-4-(3H)-one D,Llysine salt,5-methyl-7-n-propyl-8-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-[1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one,2,7-diethyl-5-[[2′-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]triazolepotassium salt,2-[2-butyl-4,5-dihydro-4-oxo-3-[2′-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3H-imidazol[4,5-c]pyridine-5-ylmethyl]benzoicacid, ethyl ester, potassium salt,3-methoxy-2,6-dimethyl-4-[[2′(1H-tetrazol-5-yl)-1,1′-biphenyl-4-yl]methoxy]pyridine,2-ethoxy-1-[[2′-(5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylicacid,1-[N-(2′-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)cyclopentane-1-carboxylicacid,7-methyl-2n-propyl-3-[[2′1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-6]pyridine,2-[5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quinolinylisodiumbenzoate,2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]pyridine,2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]benzoicacid tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one,4(S)-[4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imidazol-1-yl]octanoyl]-L-proline,1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(1H-tetrazol-5-yl)phenyl]-3-pyridinyl]methyl]-2H-imidazol-2-one,5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2′(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalene-9-one,4-[1-[2′-(1,2,3,4-tetrazol-5-yl)biphen-4-yl)methylamino]-5,6,7,8-tetrahydro-2-trifylquinazoline,2-(2-chlorobenzoyl)imino-5-ethyl-3-[2′-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl-1,3,4-thiadiazoline,2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-1,3,4-thiazoline-2-ylidene]aminocarbonyl-1-cyclopentencarboxylicacid dipotassium salt, and2-butyl-4-[N-methyl-N-(3-methylcrotonoyl)amino]-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidzole-5-carboxylicacid 1-ethoxycarbonyloxyethyl ester, those disclosed in patentpublications EP475206, EP497150, EP539086, EP539713, EP535463, EP535465,EP542059, EP497121, EP535420, EP407342, EP415886, EP424317, EP435827,EP433983, EP475898, EP490820, EP528762, EP324377, EP323841, EP420237,EP500297, EP426021, EP480204, EP429257, EP430709, EP434249, EP446062,EP505954, EP524217, EP514197, EP514198, EP514193, EP514192, EP450566,EP468372, EP485929, EP503162, EP533058, EP467207 EP399731, EP399732,EP412848, EP453210, EP456442, EP470794, EP470795, EP495626, EP495627,EP499414, EP499416, EP499415, EP511791, EP516392, EP520723, EP520724,EP539066, EP438869, EP505893, EP530702, EP400835, EP400974, EP401030,EP407102, EP411766, EP409332, EP412594, EP419048, EP480659, EP481614,EP490587, EP467715, EP479479, EP502725, EP503838, EP505098, EP505111EP513,979 EP507594, EP510812, EP511767, EP512675, EP512676, EP512870,EP517357, EP537937, EP534706, EP527534, EP540356, EP461040, EP540039,EP465368, EP498723, EP498722, EP498721, EP515265, EP503785, EP501892,EP519831, EP532410, EP498361, EP432737, EP504888, EP508393, EP508445,EP403159, EP403158, EP425211, EP427463, EP437103, EP481448, EP488532,EP501269, EP500409, EP540400, EP005528, EP028834, EP028833, EP411507,EP425921, EP430300, EP434038, EP442473, EP443568, EP445811, EP459136,EP483683, EP518033, EP520423, EP531876, EP531874, EP392317, EP468470,EP470543, EP502314, EP529253, EP543263, EP540209, EP449699, EP465323,EP521768, EP415594, WO92/14468, WO93/08171, WO93/08169, WO91/00277,WO91/00281, WO91/14367, WO92/00067, WO92/00977, WO92/20342, WO93/04045,WO93/04046, WO91/15206, WO92/14714, WO92/09600, WO92/16552, WO93/05025,WO93/03018, WO91/07404, WO92/02508, WO92/13853, WO91/19697, WO91/11909,WO91/12001, WO91/11999, WO91/15209, WO91/15479, WO92/20687, WO92/20662,WO92/20661, WO93/01177, WO91/14679, WO91/13063, WO92/13564, WO91/17148,WO91/18888, WO91/19715, WO92/02257, WO92/04335, WO92/05161, WO92/07852,WO92/15577, WO93/03033, WO91/16313, WO92/00068, WO92/02510, WO92/09278,WO9210179, WO92/10180, WO92/10186, WO92/10181, WO92/10097, WO92/10183,WO92/10182, WO92/10187, WO92/10184, WO92/10188, WO92/10180, WO92/10185,WO92/20651, WO93/03722, WO93/06828, WO93/03040, WO92/19211, WO92/22533,WO92/06081, WO92/05784, WO93/00341, WO92/04343, WO92/04059, U.S. Pat.No. 5,104,877, U.S. Pat. No. 5,187,168, U.S. Pat. No. 5,149,699, U.S.Pat. No. 5,185,340, U.S. Pat. No. 4,880,804, U.S. Pat. No. 5,138,069,U.S. Pat. No. 4,916,129, U.S. Pat. No. 5,153,197, U.S. Pat. No.5,173,494, U.S. Pat. No. 5,137,906, U.S. Pat. No. 5,155,126, U.S. Pat.No. 5,140,037, U.S. Pat. No. 5,137,902, U.S. Pat. No. 5,157,026, U.S.Pat. No. 5,053,329, U.S. Pat. No. 5,132,216, U.S. Pat. No. 5,057,522,U.S. Pat. No. 5,066586, U.S. Pat. No. 5,089,626, U.S. Pat. No.5,049,565, U.S. Pat. No. 5,087,702, U.S. Pat. No. 5,124,335, U.S. Pat.No. 5,102,880, U.S. Pat. No. 5,128,327, U.S. Pat. No. 5,151,435, U.S.Pat. No. 5,202,322, U.S. Pat. No. 5,187,159, U.S. Pat. No. 5,198,438,U.S. Pat. No. 5,182,288, U.S. Pat. No. 5,036,048, U.S. Pat. No.5,140,036, U.S. Pat. No. 5,087,634, U.S. Pat. No. 5,196,537, U.S. Pat.No. 5,153,347, U.S. Pat. No. 5,191,086, U.S. Pat. No. 5,190,942, U.S.Pat. No. 5,177,097, U.S. Pat. No. 5,212,177, U.S. Pat. No. 5,208,234,U.S. Pat. No. 5,208,235, U.S. Pat. No. 5,212,195, U.S. Pat. No.5,130,439, U.S. Pat. No. 5,045,540, U.S. Pat. No. 5,041,152, and U.S.Pat. No. 5,210,204, and pharmaceutically acceptable salts and estersthereof; α/β adrenergic blockers such as nipradilol, arotinolol,amosulalol, bretylium tosylate (CAS RN: 61-75-6), dihydroergtaminemesylate (such asergotaman-3′,6′,18-trione,9,-10-dihydro-12′-hydroxy-2′-methyl-5′-(phenylmethyl)-,(5′(a))-,monomethanesulfonate, e.g., DHE 45® Injection, Novartis), carvedilol(such as(±)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol,e.g., Coreg®, SmithKline Beecham), labetalol (such as5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)amino]ethyljsalicylamidemonohydrochloride, e.g., Normodyne®, Schering), bretylium tosylate(Benzenemethanaminium, 2-bromo-N-ethyl-N,N-dimethyl-, salt with4-methylbenzenesulfonic acid (1:1) CAS RN 61-75-6), phentolaminemesylate (Phenol,3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-,monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate(5H-1,3-Dioxolo[4,5-f]indole,7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-,(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5), zolertinehydrochloride (piperazine, 1-phenyl4-[2-(1H-tetrazol-5-yl)ethyl]-,monohydrochloride (8C1, 9C1) CAS RN 7241-94-3) and the like; αadrenergic receptor blockers, such as alfuzosin (CAS RN: 81403-68-1),terazosin, urapidil, prazosin (Minipress®), tamsulosin, bunazosin,trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XENOlO,fenspiride hydrochloride (which may be prepared as disclosed in U.S.Pat. No. 3,399,192), proroxan (CAS RN 33743-96-3), and labetalolhydrochloride and combinations thereof; a 2 agonists such as methyldopa,methyldopa HCL, lofexidine, tiamenidine, moxonidine, rilmenidine,guanobenz, and the like; aldosterone inhibitors, and the like; renininhibitors including Aliskiren (SPP100; Novartis/Speedel);angiopoietin-2-binding agents such as those disclosed in WO03/030833;anti-angina agents such as ranolazine (hydrochloride1-piperazineacetamide,N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6), betaxolol hydrochloride (2-Propanol,1-[4-[2 (cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 63659-19-8), butoprozine hydrochloride (Methanone,[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,monohydrochloride CAS RN 62134-34-3), cinepazetmaleatel-piperazineacetic acid,4-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-, ethyl ester,(2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), tosifen(Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN32295-184), verapamilhydrochloride (Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-,monohydrochloride CAS RN 152-114), molsidomine (1,2,3-Oxadiazolium,5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN25717-80-0), and ranolazine hydrochloride (1-piperazineacetamide,N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-meth-oxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN32295-184); adrenergic stimulants such as guanfacine hydrochloride (suchas N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride, e.g.,Tenex® Tablets available from Robins); methyldopa-hydrochlorothiazide(such as levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined withHydrochlorothiazide (such as6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide1,1-dioxide, e.g., the combination as, e.g., Aldoril® Tablets availablefrom Merck), methyldopa-chlorothiazide (such as6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide andmethyldopa as described above, e.g., Aldoclor®, Merck), clonidinehydrochloride (such as 2-(2,6-dichlorophenylamino)-2-imidazolinehydrochloride and chlorthalidone (such as2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl)benzenesulfonamide), e.g.,Combipres®, Boehringer Ingelheim), clonidine hydrochloride (such as2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride, e.g.,Catapres®, Boehringer Ingelheim), clonidine (1H-Imidazol-2-amine,N-(2,6-dichlorophenyl)4,5-dihydro-CAS RN 4205-90-7), Hyzaar (Merck; acombination of losartan and hydrochlorothiazide), Co-Diovan (Novartis; acombination of valsartan and hydrochlorothiazide, Lotrel (Novartis; acombination of benazepril and amlodipine) and Caduet (Pfizer; acombination of amlodipine and atorvastatin), and those agents disclosedin US20030069221.

Agents for the Treatment of Respiratory Disorders

The GCRA peptides described herein can be used in combination therapywith one or more of the following agents useful in the treatment ofrespiratory and other disorders including but not limited to: (1)β-agonists including but not limited to: albuterol (PRO VENTIL®, S ALBUTAMOK), VENTOLIN®), bambuterol, bitoterol, clenbuterol, fenoterol,formoterol, isoetharine (BRONKOSOL®, BRONKOMETER®), metaproterenol(ALUPENT®, METAPREL®), pirbuterol (MAXAIR®), reproterol, rimiterol,salmeterol, terbutaline (BRETHAIRE®, BRETHINE®, BRICANYL®), adrenalin,isoproterenol (ISUPREL®), epinephrine bitartrate (PRIMATENE®),ephedrine, orciprenline, fenoterol and isoetharine; (2) steroids,including but not limited to beclomethasone, beclomethasonedipropionate, betamethasone, budesonide, bunedoside, butixocort,dexamethasone, flunisolide, fluocortin, fluticasone, hydrocortisone,methyl prednisone, mometasone, predonisolone, predonisone, tipredane,tixocortal, triamcinolone, and triamcinolone acetonide; (3)β2-agonist-corticosteroid combinations [e.g., salmeterol-fluticasone (ADV AIR®), formoterol-budesonid (S YMBICORT®)]; (4) leukotriene D4receptor antagonists/leukotriene antagonists/LTD4 antagonists (i.e., anycompound that is capable of blocking, inhibiting, reducing or otherwiseinterrupting the interaction between leukotrienes and the Cys LTIreceptor) including but not limited to: zafhiukast, montelukast,montelukast sodium (SINGULAIR®), pranlukast, iralukast, pobilukast,SKB-106,203 and compounds described as having LTD4 antagonizing activitydescribed in U.S. Pat. No. 5,565,473; (5) 5-lipoxygenase inhibitorsand/or leukotriene biosynthesis inhibitors [e.g., zileuton and BAY1005(CA registry 128253-31-6)]; (6) histamine H1 receptorantagonists/antihistamines (i.e., any compound that is capable ofblocking, inhibiting, reducing or otherwise interrupting the interactionbetween histamine and its receptor) including but not limited to:astemizole, acrivastine, antazoline, azatadine, azelastine, astamizole,bromopheniramine, bromopheniramine maleate, carbinoxamine, carebastine,cetirizine, chlorpheniramine, chloropheniramine maleate, cimetidineclemastine, cyclizine, cyproheptadine, descarboethoxyloratadine,dexchlorpheniramine, dimethindene, diphenhydramine, diphenylpyraline,doxylamine succinate, doxylamine, ebastine, efletirizine, epinastine,famotidine, fexofenadine, hydroxyzine, hydroxyzine, ketotifen,levocabastine, levocetirizine, levocetirizine, loratadine, meclizine,mepyramine, mequitazine, methdilazine, mianserin, mizolastine,noberastine, norasternizole, noraztemizole, phenindamine, pheniramine,picumast, promethazine, pynlamine, pyrilamine, ranitidine, temelastine,terfenadine, trimeprazine, tripelenamine, and triprolidine; (7) ananticholinergic including but not limited to: atropine, benztropine,biperiden, flutropium, hyoscyamine (e.g. Levsin®; Levbid®; Levsin/SL®,Anaspaz®, Levsinex Timecaps®, NuLev®), ilutropium, ipratropium,ipratropium bromide, methscopolamine, oxybutinin, rispenzepine,scopolamine, and tiotropium; (8) an anti-tussive including but notlimited to: dextromethorphan, codeine, and hydromorphone; (9) adecongestant including but not limited to: pseudoephedrine andphenylpropanolamine; (10) an expectorant including but not limited to:guafenesin, guaicolsulfate, terpin, ammonium chloride, glycerolguaicolate, and iodinated glycerol; (11) a bronchodilator including butnot limited to: theophylline and aminophylline; (12) ananti-inflammatory including but not limited to: fluribiprofen,diclophenac, indomethacin, ketoprofen, S-ketroprophen, tenoxicam; (13) aPDE (phosphodiesterase) inhibitor including but not limited to thosedisclosed herein; (14) a recombinant humanized monoclonal antibody [e.g.xolair (also called omalizumab), rhuMab, and talizumab]; (15) ahumanized lung surfactant including recombinant forms of surfactantproteins SP-B, SP-C or SP-D [e.g. SURFAXIN®, formerly known as dsc-104(Discovery Laboratories)], (16) agents that inhibit epithelial sodiumchannels (ENaC) such as amiloride and related compounds; (17)antimicrobial agents used to treat pulmonary infections such asacyclovir, amikacin, amoxicillin, doxycycline, trimethoprinsulfamethoxazole, amphotericin B, azithromycin, clarithromycin,roxithromycin, clarithromycin, cephalosporins(ceffoxitin, cefinetazoleetc), ciprofloxacin, ethambutol, gentimycin, ganciclovir, imipenem,isoniazid, itraconazole, penicillin, ribavirin, rifampin, rifabutin,amantadine, rimantidine, streptomycin, tobramycin, and vancomycin; (18)agents that activate chloride secretion through Ca++ dependent chloridechannels (such as purinergic receptor (P2Y(2) agonists); (19) agentsthat decrease sputum viscosity, such as human recombinant DNase 1,(Pulmozyme®); (20) nonsteroidal anti-inflammatory agents (acemetacin,acetaminophen, acetyl salicylic acid, alclofenac, alminoprofen, apazone,aspirin, benoxaprofen, bezpiperylon, bucloxic acid, carprofen, clidanac,diclofenac, diclofenac, diflunisal, diflusinal, etodolac, fenbufen,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, flufenisal, fluprofen, flurbiprofen,flurbiprofen, furofenac, ibufenac, ibuprofen, indomethacin,indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen,ketorolac, meclofenamic acid, meclofenamic acid, mefenamic acid,mefenamic acid, miroprofen, mofebutazone, nabumetone oxaprozin,naproxen, naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone,phenacetin, phenylbutazone, phenylbutazone, piroxicam, piroxicam,pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine, sulindac,sulindac, suprofen, tiaprofenic acid, tiopinac, tioxaprofen, tolfenamicacid, tolmetin, tolmetin, zidometacin, zomepirac, and zomepirac); and(21) aerosolized antioxidant therapeutics such as S-Nitrosoglutathione.

Anti-Obesity Agents

The GCRA peptides described herein can be used in combination therapywith an anti-obesity agent. Suitable such agents include, but are notlimited to: 1 1β HSD-I (11-beta hydroxy steroid dehydrogenase type 1)inhibitors, such as BVT 3498, BVT 2733,3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,3-(1-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][ll]annulene, and those compounds disclosed in WO01/90091, WO 01/90090, WO01/90092 and WO02/072084; 5HT antagonists such as those in WO03/037871,WO03/037887, and the like; SHTIa modulators such as carbidopa,benserazide and those disclosed in U.S. Pat. No. 6,207,699, WO03/031439,and the like; 5HT2c (serotonin receptor 2c) agonists, such as BVT933,DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213 (Glaxo SmithKline) and YM 348 and those disclosed in U.S. Pat. No. 3,914,250,WO00/77010, WO02/36596, WO02/48124, WO02/10169, WO01/66548, WO02/44152,WO02/51844, WO02/40456, and WO02/40457; 5HT6 receptor modulators, suchas those in WO03/030901, WO03/035061, WO03/039547, and the like;acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa, M.et al, Obesity Research, 9:202-9 (2001) and Japanese Patent ApplicationNo. JP 2000256190; anorectic bicyclic compounds such as 1426 (Aventis)and 1954 (Aventis), and the compounds disclosed in WO00/18749,WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB 1 (cannabinoid-1receptor) antagonist/inverse agonists such as rimonabant (Acomplia;Sanofi), SR-147778 (Sanofi), SR-141716 (Sanofi), BAY 65-2520 (Bayer),and SLV 319 (Solvay), and those disclosed in patent publications U.S.Pat. No. 4,973,587, U.S. Pat. No. 5,013,837, U.S. Pat. No. 5,081,122,U.S. Pat. No. 5,112,820, U.S. Pat. No. 5,292,736, U.S. Pat. No.5,532,237, U.S. Pat. No. 5,624,941, U.S. Pat. No. 6,028,084, U.S. Pat.No. 6,509,367, U.S. Pat. No. 6,509,367, WO96/33159, WO97/29079,WO98/31227, WO98/33765, WO98/37061, WO98/41519, WO98/43635, WO98/43636,WO99/02499, WO00/10967, WO00/10968, WO01/09120, WO01/58869, WO01/64632,WO01/64633, WO01/64634, WO01/70700, WO01/96330, WO02/076949,WO03/006007, WO03/007887, WO03/020217, WO03/026647, WO03/026648,WO03/027069, WO03/027076, WO03/027114, WO03/037332, WO03/040107,WO03/086940, WO03/084943 and EP658546; CCK-A (cholecystokinin-A)agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, A-71378, A-71623and SR146131 (Sanofi), and those described in U.S. Pat. No. 5,739,106;CNTF (Ciliary neurotrophic factors), such as GI-181771(Glaxo-SmithKline), SR146131 (Sanofi Synthelabo), butabindide, PD170,292, and PD 149164 (Pfizer); CNTF derivatives, such as Axokine®(Regeneron), and those disclosed in WO94/09134, WO98/22128, andWO99/43813; dipeptidyl peptidase IV (DP-IV) inhibitors, such asisoleucine thiazolidide, valine pyrrolidide, NVP-DPP728, LAF237, P93/01,P 3298, TSL 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid; disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)1537-1540), TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364, VIP0177, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides asdisclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22,pp 1163-1166 and 2745-2748 (1996) and the compounds disclosed patentpublications. WO99/38501, WO99/46272, WO99/67279 (Probiodrug),WO99/67278 (Probiodrug), WO99/61431 (Probiodrug), WO02/083128,WO02/062764, WO03/000180, WO03/000181, WO03/000250, WO03/002530,WO03/002531, WO03/002553, WO03/002593, WO03/004498, WO03/004496,WO03/017936, WO03/024942, WO03/024965, WO03/033524, WO03/037327 andEP1258476; growth hormone secretagogue receptor agonists/antagonists,such as NN703, hexarelin, MK-0677 (Merck), SM-130686, CP-424391(Pfizer), LY 444,711 (Eli Lilly), L-692,429 and L-163,255, and such asthose disclosed in U.S. Ser. No. 09/662,448, U.S. provisionalapplication 60/203,335, U.S. Pat. No. 6,358,951, US2002049196,US2002/022637, WO01/56592 and WO02/32888; H3 (histamine H3)antagonist/inverse agonists, such as thioperamide,3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit,iodophenpropit, imoproxifan, GT2394 (Gliatech), and A331440,O-[3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz, K. et al.,Pharmazie, 55:349-55 (2000)), piperidine-containing histamineH3-receptor antagonists (Lazewska, D. et al., Pharmazie, 56:927-32(2001), benzophenone derivatives and related compounds (Sasse, A. etal., Arch. Pharm. (Weinheim) 334:45-52 (2001)), substitutedN-phenylcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)),and proxifan derivatives (Sasse, A. et al., J. Med. Chem. 43:3335-43(2000)) and histamine H3 receptor modulators such as those disclosed inWO02/15905, WO03/024928 and WO03/024929; leptin derivatives, such asthose disclosed in U.S. Pat. No. 5,552,524, U.S. Pat. No. 5,552,523,U.S. Pat. No. 5,552,522, U.S. Pat. No. 5,521,283, WO96/23513,WO96/23514, WO96/23515, WO96/23516, WO96/23517, WO96/23518, WO96/23519,and WO96/23520; leptin, including recombinant human leptin (PEG-OB,Hoffman La Roche) and recombinant methionyl human leptin (Amgen); lipaseinhibitors, such as tetrahydrolipstatin (orlistat/Xenical®), Triton WR1339, RHC80267, lipstatin, teasaponin, diethylumbelliferyl phosphate,FL-386, WAY-121898, Bay-N-3176, valilactone, esteracin, ebelactone A,ebelactone B, and RHC 80267, and those disclosed in patent publicationsWO01/77094, U.S. Pat. No. 4,598,089, U.S. Pat. No. 4,452,813, U.S. Pat.No. 5,512,565, U.S. Pat. No. 5,391,571, U.S. Pat. No. 5,602,151, U.S.Pat. No. 4,405,644, U.S. Pat. No. 4,189,438, and U.S. Pat. No.4,242,453; lipid metabolism modulators such as maslinic acid,erythrodiol, ursolic acid uvaol, betulinic acid, betulin, and the likeand compounds disclosed in WO03/011267; Mc4r (melanocortin 4 receptor)agonists, such as CHIR86036 (Chiron), ME-10142, ME-10145, and HS-131(Melacure), and those disclosed in PCT publication Nos. WO99/64002,WO00/74679, WO 01/991752, WO 01/25192, WO 01/52880, WO 01/74844, WO01/70708, WO01/70337, WO01/91752, WO02/059095, WO02/059107, WO02/059108,WO02/059117, WO02/06276, WO02/12166, WO02/11715, WO02/12178, WO02/15909,WO02/38544, WO02/068387, WO02/068388, WO02/067869, WO02/081430,WO03/06604, WO03/007949, WO03/009847, WO03/009850, WO03/013509, andWO03/031410; Mc5r (melanocortin 5 receptor) modulators, such as thosedisclosed in WO97/19952, WO00/15826, WO00/15790, US20030092041;melanin-concentrating hormone 1 receptor (MCHR) antagonists, such asT-226296 (Takeda), SB 568849, SNP-7941 (Synaptic), and those disclosedin patent publications WO 01/21169, WO01/82925, WO01/87834, WO02/051809,WO02/06245, WO02/076929, WO02/076947, WO02/04433, WO02/51809,WO02/083134, WO02/094799, WO03/004027, WO03/13574, WO03/15769,WO03/028641, WO03/035624, WO03/033476, WO03/033480, JP13226269, andJP1437059; mGluR5 modulators such as those disclosed in WO03/029210,WO03/047581, WO03/048137, WO03/051315, WO03/051833, WO03/053922,WO03/059904, and the like; serotoninergic agents, such as fenfluramine(such as Pondimin® (Benzeneethanamine,N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Robbins),dexfenfluramine (such as Redux® (Benzeneethanamine,N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Interneuron)and sibutramine ((Meridia®, Knoll/Reductil™) including racemic mixtures,as optically pure isomers (+) and (−), and pharmaceutically acceptablesalts, solvents, hydrates, clathrates and prodrugs thereof includingsibutramine hydrochloride monohydrate salts thereof, and those compoundsdisclosed in U.S. Pat. No. 4,746,680, U.S. Pat. No. 4,806,570, and U.S.Pat. No. 5,436,272, US20020006964, WO 01/27068, and WO 01/62341; NE(norepinephrine) transport inhibitors, such as GW 320659, despiramine,talsupram, and nomifensine; NPY 1 antagonists, such as BIBP3226,J-115814, BIBO 3304, LY-357897, CP-671906, GI-264879A, and thosedisclosed in U.S. Pat. No. 6,001,836, WO96/14307, WO01/23387,WO99/51600, WO01/85690, WO01/85098, WO01/85173, and WO01/89528; NPYS(neuropeptide Y Y5) antagonists, such as 152,804, GW-569180A,GW-594884A, GW-587081×, GW-548118×, FR235208, FR226928, FR240662,FR252384, 1229U91, GI-264879A, CGP71683A, LY-377897, LY-366377,PD-160170, SR-120562A, SR-120819A, JCF-104, and H409/22 and thosecompounds disclosed in patent publications U.S. Pat. No. 6,140,354, U.S.Pat. No. 6,191,160, U.S. Pat. No. 6,218,408, U.S. Pat. No. 6,258,837,U.S. Pat. No. 6,313,298, U.S. Pat. No. 6,326,375, U.S. Pat. No.6,329,395, U.S. Pat. No. 6,335,345, U.S. Pat. No. 6,337,332, U.S. Pat.No. 6,329,395, U.S. Pat. No. 6,340,683, EP01010691, EP-01044970,WO97/19682, WO97/20820, WO97/20821, WO97/20822, WO97/20823, WO98/27063,WO00/107409, WO00/185714, WO00/185730, WO00/64880, WO00/68197,WO00/69849, WO/0113917, WO01/09120, WO01/14376, WO01/85714, WO01/85730,WO01/07409, WO01/02379, WO01/23388, WO01/23389, WO 01/44201, WO01/62737,WO01/62738, WO01/09120, WO02/20488, WO02/22592, WO02/48152, WO02/49648,WO02/051806, WO02/094789, WO03/009845, WO03/014083, WO03/022849,WO03/028726 and Norman et al, J. Med. Chem. 43:4288-4312 (2000); opioidantagonists, such as nalmefene (REVEX®), 3-methoxynaltrexone,methylnaltrexone, naloxone, and naltrexone (e.g. PT901; PainTherapeutics, Inc.) and those disclosed in US20050004155 and WO00/21509;orexin antagonists, such as SB-334867-A and those disclosed in patentpublications WO01/96302, WO01/68609, WO02/44172, WO02/51232, WO02/51838,WO02/089800, WO02/090355, WO03/023561, WO03/032991, and WO03/037847; PDEinhibitors (e.g. compounds which slow the degradation of cyclic AMP(cAMP) and/or cyclic GMP (cGMP) by inhibition of the phosphodiesterases,which can lead to a relative increase in the intracellular concentrationof cAMP and cGMP; possible PDE inhibitors are primarily those substanceswhich are to be numbered among the class consisting of the PDE3inhibitors, the class consisting of the PDE4 inhibitors and/or the classconsisting of the PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors) such as those disclosed in patent publicationsDE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,EP0075436, EP0096517, EPOl 12987, EPOl 16948, EP0150937, EP0158380,EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811,EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117,EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,JP92234389, JP94329652, JP95010875, U.S. Pat. No. 4,963,561, U.S. Pat.No. 5,141,931, WO9117991, WO9200968, WO9212961, WO9307146, WO9315044,WO9315045, WO9318024, WO9319068, WO9319720, WO9319747, WO9319749,WO9319751, WO9325517, WO9402465, WO9406423, WO9412461, WO9420455,WO9422852, WO9425437, WO9427947, WO9500516, WO9501980, WO9503794,WO9504045, WO9504046, WO9505386, WO9508534, WO9509623, WO9509624,WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392,WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, WO9528926,WO9535281, WO9535282, WO9600218, WO9601825, WO9602541, WO9611917,DE3142982, DE1 116676, DE2162096, EP0293063, EP0463756, EP0482208,EP0579496, EP0667345 U.S. Pat. No. 6,331,543, US20050004222 (includingthose disclosed in formulas I-XIII and paragraphs 37-39, 85-0545 and557-577), WO9307124, EP0163965, EP0393500, EP0510562, EP0553174,WO9501338 and WO9603399, as well as PDE5 inhibitors (such as RX-RA-69,SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra™)), PDE4 inhibitors (such asetazolate, ICI63197, RP73401, imazolidinone (RO-20-1724), MEM 1414(R1533/R1500; Pharmacia Roche), denbufylline, rolipram, oxagrelate,nitraquazone, Y-590, DH-6471, SKF-94120, motapizone, lixazinone,indolidan, olprinone, atizoram, KS-506-G, dipamfylline, BMY-43351,atizoram, arofylline, filaminast, PDB-093, UCB-29646, CDP-840,SKF-107806, piclamilast, RS-17597, RS-25344-000, SB-207499, TIBENELAST,SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840,mopidamol, anagrelide, ibudilast, aminone, pimobendan, cilostazol,quazinone andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamide,PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867), MCI-154,UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran, piroximone,imazodan, CI-930, siguazodan, adibendan, saterinone, SKF-95654,SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307,revizinone, NM-702, WIN-62582 and WIN-63291, enoximone and milrinone,PDE3/4 inhibitors (such as benafentrine, trequinsin, ORG-30029,zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andtolafentrine) and other PDE inhibitors (such as vinpocetin, papaverine,enprofylline, cilomilast, fenoximone, pentoxifylline, roflumilast,tadalafil(Clalis®), theophylline, and vardenafil(Levitra®); NeuropeptideY2 (NPY2) agonists include but are not limited to: polypeptide YY andfragments and variants thereof (e.g. YY3-36 (PYY3-36)(N. Engl. J. Med.349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO:107))and PYY agonists such as those disclosed in WO02/47712, WO03/026591,WO03/057235, and WO03/027637; serotonin reuptake inhibitors, such as,paroxetine, fluoxetine (Prozac™), fluvoxamine, sertraline, citalopram,and imipramine, and those disclosed in U.S. Pat. No. 6,162,805, U.S.Pat. No. 6,365,633, WO03/00663, WO 01/27060, and WO 01/162341; thyroidhormone β agonists, such as KB-2611 (KaroBioBMS), and those disclosed inWO02/15845, WO97/21993, WO99/00353, GB98/284425, U.S. ProvisionalApplication No. 60/183,223, and Japanese Patent Application No. JP2000256190; UCP-I (uncoupling protein-1), 2, or 3 activators, such asphytanic acid,4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoicacid (TTNPB), retinoic acid, and those disclosed in WO99/00123; P3 (betaadrenergic receptor 3) agonists, such as AJ9677/TAK677(Dainippon/Takeda), L750355 (Merck), CP331648 (Pfizer), CL-316,243, SB418790, BRL-37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243,GW 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604(Lilly), SR 59119A, and those disclosed in U.S. Pat. No. 5,541,204, U.S.Pat. No. 5,770,615, U.S. Pat. No. 5,491,134, U.S. Pat. No. 5,776,983,U.S. Pat. No. 488,064, U.S. Pat. No. 5,705,515, U.S. Pat. No. 5,451,677,WO94/18161, WO95/29159, WO97/46556, WO98/04526 and WO98/32753,WO01/74782, WO02/32897, WO03/014113, WO03/016276, WO03/016307,WO03/024948, WO03/024953 and WO03/037881; noradrenergic agentsincluding, but not limited to, diethylpropion (such as Tenuate®(1-propanone, 2-(diethylamino)-1-phenyl-, hydrochloride), Merrell),dextroamphetamine (also known as dextroamphetamine sulfate,dexamphetamine, dexedrine, Dexampex, Ferndex, Oxydess II, Robese,Spancap #1), mazindol ((or5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol) such asSanorex®, Novartis or Mazanor®, Wyeth Ayerst), phenylpropanolamine (orBenzenemethanol, alpha-(1-aminoethyl)-, hydrochloride), phentermine ((orPhenol, 3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino],monohydrochloride) such as Adipex-P®, Lemmon, FASTIN®, Smith-KlineBeecham and Ionamin®, Medeva), phendimetrazine ((or(2S,3S)-3,4-Dimethyl-2-phenylmorpholine L-(+)-tartrate (1:1)) such asMetra® (Forest), Plegine® (Wyeth-Ay erst), Prelu-2® (BoehringerIngelheim), and Statobex® (Lemmon), phendamine tartrate (such asThephorin®(2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridineL-(+)-tartrate (1:1)), Hoffmann-LaRoche), methamphetamine (such asDesoxyn®, Abbot ((S)—N, (alpha)-dimethylbenzeneethanaminehydrochloride)), and phendimetrazine tartrate (such as Bontril®Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholineTartrate); fatty acid oxidation upregulator/inducers such as Famoxin®(Genset); monamine oxidase inhibitors including but not limited tobefloxatone, moclobemide, brofaromine, phenoxathine, esuprone, befol,toloxatone, pirlindol, amiflamine, sercloremine, bazinaprine,lazabemide, milacemide, caroxazone and other certain compounds asdisclosed by WO01/12176; and other anti-obesity agents such as 5HT-2agonists, ACC (acetyl-CoA carboxylase) inhibitors such as thosedescribed in WO03/072197, alpha-lipoic acid (alpha-LA), AOD9604,appetite suppressants such as those in WO03/40107, ATL-962 (AlizymePLC), benzocaine, benzphetamine hydrochloride (Didrex), bladderwrack(focus vesiculosus), BRS3 (bombesin receptor subtype 3) agonists,bupropion, caffeine, CCK agonists, chitosan, chromium, conjugatedlinoleic acid, corticotropin-releasing hormone agonists,dehydroepiandrosterone, DGAT1 (diacylglycerol acyltransferase 1)inhibitors, DGAT2 (diacylglycerol acyltransferase 2) inhibitors,dicarboxylate transporter inhibitors, ephedra, exendin-4 (an inhibitorof glp-1) FAS (fatty acid synthase) inhibitors (such as Cerulenin andC75), fat resorption inhibitors (such as those in WO03/053451, and thelike), fatty acid transporter inhibitors, natural water soluble fibers(such as psyllium, plantago, guar, oat, pectin), galanin antagonists,galega (Goat's Rue, French Lilac), garcinia cambogia, germander(teucrium chamaedrys), ghrelin antibodies and ghrelin antagonists (suchas those disclosed in WO01/87335, and WO02/08250), polypeptide hormonesand variants thereof which affect the islet cell secretion, such as thehormones of the secretin/gastric inhibitory polypeptide (GIP)/vasoactiveintestinal polypeptide (VIP)/pituitary adenylate cyclase activatingpolypeptide (PACAP)/glucagon-like polypeptide II(GLP-II)/glicentin/glucagon gene family and/or those of theadrenomedullin/amylin/calcitonin gene related polypeptide (CGRP) genefamily includingGLP-1 (glucagon-like polypeptide 1) agonists (e.g. (1)exendin-4, (2) those GLP-1 molecules described in US20050130891including GLP-1(7-34), GLP-1(7-35), GLP-1(7-36) or GLP-1(7-37) in itsC-terminally carboxylated or amidated form or as modified GLP-Ipolypeptides and modifications thereof including those described inparagraphs 17-44 of US20050130891, and derivatives derived fromGLP-1-(7-34)COOH and the corresponding acid amide are employed whichhave the following general formula:R—NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH₂ (SEQ ID NO: 108) wherein R═H oran organic compound having from 1 to 10 carbon atoms. Preferably, R isthe residue of a carboxylic acid. Particularly preferred are thefollowing carboxylic acid residues: formyl, acetyl, propionyl,isopropionyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl.) and glp-1 (glucagon-like polypeptide-1), glucocorticoidantagonists, glucose transporter inhibitors, growth hormonesecretagogues (such as those disclosed and specifically described inU.S. Pat. No. 5,536,716), interleukin-6 (IL-6) and modulators thereof(as in WO03/057237, and the like), L-carnitine, Mc3r (melanocortin 3receptor) agonists, MCH2R (melanin concentrating hormone 2R)agonist/antagonists, melanin concentrating hormone antagonists,melanocortin agonists (such as Melanotan II or those described in WO99/64002 and WO 00/74679), nomame herba, phosphate transporterinhibitors, phytopharm compound 57 (CP 644,673), pyruvate, SCD-I(stearoyl-CoA desaturase-1) inhibitors, T71 (Tularik, Inc., BoulderColo.), Topiramate (Topimax®, indicated as an anti-convulsant which hasbeen shown to increase weight loss), transcription factor modulators(such as those disclosed in WO03/026576), β-hydroxy steroiddehydrogenase-1 inhibitors ((3-HSD-I), β-hydroxy-β-methylbutyrate, p57(Pfizer), Zonisamide (Zonegran™, indicated as an anti-epileptic whichhas been shown to lead to weight loss), and the agents disclosed inUS20030119428 paragraphs 20-26.

Anti-Diabetic Agents

The GCRA peptides described herein can be used in therapeuticcombination with one or more anti-diabetic agents, including but notlimited to: PPARγ agonists such as glitazones (e.g., WAY-120,744, AD5075, balaglitazone, ciglitazone, darglitazone (CP-86325, Pfizer),englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555(Mitsibishi disclosed in U.S. Pat. No. 5,594,016), pioglitazone (such assuch as Actos™ pioglitazone; Takeda), rosiglitazone (Avandia™; SmithKline Beecham), rosiglitazone maleate, troglitazone (Rezulin®, disclosedin U.S. Pat. No. 4,572,912), rivoglitazone (CS-Ol 1, Sankyo), GL-262570(Glaxo Welcome), BRL49653 (disclosed in WO98/05331), CLX-0921, 5-BTZD,GW-0207, LG-100641, JJT-501 (JPNT/P&U), L-895645 (Merck), R-119702(Sankyo/Pfizer), NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512,LY-519818, R483 (Roche), T131 (Tularik), and the like and compoundsdisclosed in U.S. Pat. No. 4,687,777, U.S. Pat. No. 5,002,953, U.S. Pat.No. 5,741,803, U.S. Pat. No. 5,965,584, U.S. Pat. No. 6,150,383, U.S.Pat. No. 6,150,384, U.S. Pat. No. 6,166,042, U.S. Pat. No. 6,166,043,U.S. Pat. No. 6,172,090, U.S. Pat. No. 6,211,205, U.S. Pat. No.6,271,243, U.S. Pat. No. 6,288,095, U.S. Pat. No. 6,303,640, U.S. Pat.No. 6,329,404, U.S. Pat. No. 5,994,554, WO97/10813, WO97/27857,WO97/28115, WO97/28137, WO97/27847, WO00/76488, WO03/000685,WO03/027112, WO03/035602, WO03/048130, WO03/055867, and pharmaceuticallyacceptable salts thereof; biguanides such as metformin hydrochloride(N,N-dimethylimidodicarbonimidic diamide hydrochloride, such asGlucophage™, Bristol-Myers Squibb); metformin hydrochloride withglyburide, such as Glucovance™, Bristol-Myers Squibb); buformin(Imidodicarbonimidic diamide, N-butyl-); etoformine(1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms(including where the salt is chosen from the group of, acetate,benzoate, citrate, ftimarate, embonate, chlorophenoxyacetate, glycolate,palmoate, aspartate, methanesulphonate, maleate,parachlorophenoxyisobutyrate, formate, lactate, succinate, sulphate,tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate,hexadecanoate, octodecanoate, benzenesulphonate, trimethoxybenzoate,paratoluenesulphonate, adamantanecarboxylate, glycoxylate, glutamate,pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,nitrate, sulphite, dithionate and phosphate), and phenformin; proteintyrosine phosphatase-IB (PTP-IB) inhibitors, such as A-401,674, KR61639, OC-060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715,and those disclosed in WO99/585521, WO99/58518, WO99/58522, WO99/61435,WO03/032916, WO03/032982, WO03/041729, WO03/055883, WO02/26707,WO02/26743, JP2002114768, and pharmaceutically acceptable salts andesters thereof; sulfonylureas such as acetohexamide (e.g. Dymelor, EliLilly), carbutamide, chlorpropamide (e.g. Diabinese®, Pfizer),gliamilide (Pfizer), gliclazide (e.g. Diamcron, Servier Canada Inc),glimepiride (e.g. disclosed in U.S. Pat. No. 4,379,785, such as Amaryl,Aventis), glipentide, glipizide (e.g. Glucotrol or Glucotrol XL ExtendedRelease, Pfizer), gliquidone, glisolamide, glyburide/glibenclamide (e.g.Micronase or Glynase Prestab, Pharmacia & Upjohn and Diabeta, Aventis),tolazamide (e.g. Tolinase), and tolbutamide (e.g. Orinase), andpharmaceutically acceptable salts and esters thereof; meglitinides suchas repaglinide (e.g. Pranidin®, Novo Nordisk), KAD1229 (PF/Kissei), andnateglinide (e.g. Starlix®, Novartis), and pharmaceutically acceptablesalts and esters thereof; a glucoside hydrolase inhibitors (or glucosideinhibitors) such as acarbose (e.g. Precose™, Bayer disclosed in U.S.Pat. No. 4,904,769), miglitol (such as GLYSETT™, Pharmacia & Upjohndisclosed in U.S. Pat. No. 4,639,436), camiglibose (Methyl6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-D-glucopyranoside,Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate,pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR 14,and the compounds disclosed in U.S. Pat. No. 4,062,950, U.S. Pat. No.4,174,439, U.S. Pat. No. 4,254,256, U.S. Pat. No. 4,701,559, U.S. Pat.No. 4,639,436, U.S. Pat. No. 5,192,772, U.S. Pat. No. 4,634,765, U.S.Pat. No. 5,157,116, U.S. Pat. No. 5,504,078, U.S. Pat. No. 5,091,418,U.S. Pat. No. 5,217,877, U.S. Pat. No. 51,091 and WO 01/47528(polyamines); α-amylase inhibitors such as tendamistat, trestatin, andAl-3688, and the compounds disclosed in U.S. Pat. No. 4,451,455, U.S.Pat. No. 4,623,714, and U.S. Pat. No. 4,273,765; SGLT2 inhibitorsincluding those disclosed in U.S. Pat. No. 6,414,126 and U.S. Pat. No.6,515,117; an aP2 inhibitor such as disclosed in U.S. Pat. No.6,548,529; insulin secreatagogues such as linogliride, A-4166,forskilin, dibutyrl cAMP, isobutylmethylxanthine (IBMX), andpharmaceutically acceptable salts and esters thereof; fatty acidoxidation inhibitors, such as clomoxir, and etomoxir, andpharmaceutically acceptable salts and esters thereof; A2 antagonists,such as midaglizole, isaglidole, deriglidole, idazoxan, earoxan, andfluparoxan, and pharmaceutically acceptable salts and esters thereof;insulin and related compounds (e.g. insulin mimetics) such as biota,LP-100, novarapid, insulin detemir, insulin lispro, insulin glargine,insulin zinc suspension (lente and ultralente), Lys-Pro insulin, GLP-I(1-36) amide, GLP-I (73-7) (insulintropin, disclosed in U.S. Pat. No.5,614,492), LY-315902 (Lilly), GLP-I (7-36)-NH2), AL-401 (Autoimmune),certain compositions as disclosed in U.S. Pat. No. 4,579,730, U.S. Pat.No. 4,849,405, U.S. Pat. No. 4,963,526, U.S. Pat. No. 5,642,868, U.S.Pat. No. 5,763,396, U.S. Pat. No. 5,824,638, U.S. Pat. No. 5,843,866,U.S. Pat. No. 6,153,632, U.S. Pat. No. 6,191,105, and WO 85/05029, andprimate, rodent, or rabbit insulin including biologically activevariants thereof including allelic variants, more preferably humaninsulin available in recombinant form (sources of human insulin includepharmaceutically acceptable and sterile formulations such as thoseavailable from Eli Lilly (Indianapolis, Ind. 46285) as Humulin™ (humaninsulin rDNA origin), also see the THE PHYSICIAN'S DESK REFERENCE,55.sup.th Ed. (2001) Medical Economics, Thomson Healthcare (disclosingother suitable human insulins); non-thiazolidinediones such as JT-501and farglitazar (GW-2570/GI-262579), and pharmaceutically acceptablesalts and esters thereof; PPARα/γ dual agonists such as AR-H039242(Aztrazeneca), GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, GW-1536,GW-1929, GW-2433, KRP-297 (Kyorin Merck; 5-[(2,4-Dioxothiazolidinyl)methyl]methoxy-N-[[4-(trifluoromethyl)phenyl]methyl]benzamide),L-796449, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar(BMS), tesaglitzar (Astrazeneca), reglitazar (JTT-501) and thosedisclosed in WO99/16758, WO99/19313, WO99/20614, WO99/38850, WO00/23415,WO00/23417, WO00/23445, WO00/50414, WO01/00579, WO01/79150, WO02/062799,WO03/004458, WO03/016265, WO03/018010, WO03/033481, WO03/033450,WO03/033453, WO03/043985, WO 031053976, U.S. application Ser. No.09/664,598, filed Sep. 18, 2000, Murakami et al. Diabetes 47, 1841-1847(1998), and pharmaceutically acceptable salts and esters thereof; otherinsulin sensitizing drugs; VPAC2 receptor agonists; GLK modulators, suchas those disclosed in WO03/015774; retinoid modulators such as thosedisclosed in WO03/000249; GSK 3β/GSK 3 inhibitors such as4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine andthose compounds disclosed in WO03/024447, WO03/037869, WO03/037877,WO03/037891, WO03/068773, EP1295884, EP1295885, and the like; glycogenphosphorylase (HGLPa) inhibitors such as CP-368,296, CP-316,819,BAYR3401, and compounds disclosed in WO 01/94300, WO02/20530,WO03/037864, and pharmaceutically acceptable salts or esters thereof;ATP consumption promotors such as those disclosed in WO03/007990; TRB3inhibitors; vanilloid receptor ligands such as those disclosed inWO03/049702; hypoglycemic agents such as those disclosed in WO03/015781and WO03/040114; glycogen synthase kinase 3 inhibitors such as thosedisclosed in WO03/035663 agents such as those disclosed in WO99/51225,US20030134890, WO01/24786, and WO03/059870; insulin-responsive DNAbinding protein-1 (IRDBP-I) as disclosed in WO03/057827, and the like;adenosine A2 antagonists such as those disclosed in WO03/035639,WO03/035640, and the like; PPARδ agonists such as GW 501516, GW 590735,and compounds disclosed in JP10237049 and WO02/14291; dipeptidylpeptidase IV (DP-IV) inhibitors, such as isoleucine thiazolidide,NVP-DPP728A(1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine,disclosed by Hughes et al, Biochemistry, 38(36), 11597-11603, 1999),P32/98, NVP-LAF-237, P3298, TSL225(tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, disclosedby Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998) 1537-1540), valinepyrrolidide, TMC-2A/2B/2C, CD-26 inhibitors, FE999011, P9310/K364, VIP0177, DPP4, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides asdisclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22,pp 1163-1166 and 2745-2748 (1996), and the compounds disclosed in U.S.Pat. No. 6,395,767, U.S. Pat. No. 6,573,287, U.S. Pat. No. 6,395,767(compounds disclosed include BMS-477118, BMS-471211 and BMS 538,305),WO99/38501, WO99/46272, WO99/67279, WO99/67278, WO99/61431WO03/004498,WO03/004496, EP1258476, WO02/083128, WO02/062764, WO03/000250,WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/000180, andWO03/000181; GLP-I agonists such as exendin-3 and exendin-4 (includingthe 39 aa polypeptide synthetic exendin-4 called Exenatide®), andcompounds disclosed in US2003087821 and NZ 504256, and pharmaceuticallyacceptable salts and esters thereof; peptides including amlintide andSymlin® (pramlintide acetate); and glycokinase activators such as thosedisclosed in US2002103199 (fused heteroaromatic compounds) andWO02/48106 (isoindolin-1-one-substituted propionamide compounds).

Phosphodiesterase Inhibitors

The GCRA peptides described herein can be used in combination therapywith a phosphodiesterase inhibitor. PDE inhibitors are those compoundswhich slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP (cGMP)by inhibition of the phosphodiesterases, which can lead to a relativeincrease in the intracellular concentration of c AMP and/or cGMP.Possible PDE inhibitors are primarily those substances which are to benumbered among the class consisting of the PDE3 inhibitors, the classconsisting of the PDE4 inhibitors and/or the class consisting of thePDE5 inhibitors, in particular those substances which can be designatedas mixed types of PDE3/4 inhibitors or as mixed types of PDE3/4/5inhibitors. By way of example, those PDE inhibitors may be mentionedsuch as are described and/or claimed in the following patentapplications and patents: DE1470341, DE2108438, DE2123328, DE2305339,DE2305575, DE2315801, DE2402908, DE2413935, DE2451417, DE2459090,DE2646469, DE2727481, DE2825048, DE2837161, DE2845220, DE2847621,DE2934747, DE3021792, DE3038166, DE3044568, EP000718, EP0008408,EP0010759, EP0059948, EP0075436, EP0096517, EPO1 12987, EPO1 16948,EP0150937, EP0158380, EP0161632, EP0161918, EP0167121, EP0199127,EP0220044, EP0247725, EP0258191, EP0272910, EP0272914, EP0294647,EP0300726, EP0335386, EP0357788, EP0389282, EP0406958, EP0426180,EP0428302, EP0435811, EP0470805, EP0482208, EP0490823, EP0506194,EP0511865, EP0527117, EP0626939, EP0664289, EP0671389, EP0685474,EP0685475, EP0685479, JP92234389, JP94329652, JP95010875, U.S. Pat. Nos.4,963,561, 5,141,931, WO9117991, WO9200968, WO9212961, WO9307146,WO9315044, WO9315045, WO9318024, WO9319068, WO9319720, WO9319747,WO9319749, WO9319751, WO9325517, WO9402465, WO9406423, WO9412461,WO9420455, WO9422852, WO9425437, WO9427947, WO9500516, WO9501980,WO9503794, WO9504045, WO9504046, WO9505386, WO9508534, WO9509623,WO9509624, WO9509627, WO9509836, WO9514667, WO9514680, WO9514681,WO9517392, WO9517399, WO9519362, WO9522520, WO9524381, WO9527692,WO9528926, WO9535281, WO9535282, WO9600218, WO9601825, WO9602541,WO9611917, DE3142982, DE1 116676, DE2162096, EP0293063, EP0463756,EP0482208, EP0579496, EP0667345 U.S. Pat. No. 6,331,543, US20050004222(including those disclosed in formulas I-XIII and paragraphs 37-39,85-0545 and 557-577) and WO9307124, EP0163965, EP0393500, EP0510562,EP0553174, WO9501338 and WO9603399. PDE5 inhibitors which may bementioned by way of example are RX-RA-69, SCH-51866, KT-734,vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 andsildenafil (Viagra®). PDE4 inhibitors which may be mentioned by way ofexample are RO-20-1724, MEM 1414 (R1533/R1500; Pharmacia Roche),DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE, Y-590, DH-6471,SKF-94120, MOTAPIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE, ATIZORAM,KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE, FILAMINAST,PDB-093, UCB-29646, CDP-840, SKF-107806, PICLAMILAST, RS-17597,RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572, SB-211600,SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL, ANAGRELIDE,IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL, QUAZINONE andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy-4-difluoromethoxybenzamide.PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE,AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930,SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85,EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702,WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitorswhich may be mentioned by way of example are BENAFENTRINE, TREQUINSIN,ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andTOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline,roflumilast, tadalafil(Clalis®), theophylline, and vardenafil(Levitra®),zaprinast (PDE5 specific).

Anti-Uterine Contractions Agents

The GCRA peptides described herein can be used in combination therapy(for example, in order to decrease or inhibit uterine contractions) witha tocolytic agent including but not limited to beta-adrenergic agents,magnesium sulfate, prostaglandin inhibitors, and calcium channelblockers.

Anti-Neoplastic Agents

The GCRA peptides described herein can be used in combination therapywith an antineoplastic agents including but not limited to alkylatingagents, epipodophyllotoxins, nitrosoureas, antimetabolites, vincaalkaloids, anthracycline antibiotics, nitrogen mustard agents, and thelike. Particular anti-neoplastic agents may include tamoxifen, taxol,etoposide and 5-fluorouracil.

The GCRA peptides described herein can be used in combination therapy(for example as in a chemotherapeutic composition) with an antiviral andmonoclonal antibody therapies.

Agents to Treat Congestive Heart Failure

The GCRA peptides described herein can be used in combination therapy(for example, in prevention/treatment of congestive heart failure oranother method described herein) with the partial agonist of thenociceptin receptor ORL1 described by Dooley et al. (The Journal ofPharmacology and Experimental Therapeutics, 283 (2): 735-741, 1997). Theagonist is a hexapeptide having the amino acid sequence Ac-RYY (RK) (WI)(RK)-NH2 (“the Dooley polypeptide”), where the brackets show allowablevariation of amino acid residue. Thus Dooley polypeptide can include butare not limited to KYYRWR (SEQ ID NO: 109), RYYRWR (SEQ ID NO: 110),KWRYYR (SEQ ID NO: 111), RYYRWK (SEQ ID NO: 112), RYYRWK (all-D aminacids) (SEQ ID NO: 113), RYYRIK (SEQ ID NO: 114), RYYRIR (SEQ ID NO:115), RYYKIK (SEQ ID NO: 116), RYYKIR (SEQ ID NO: 117), RYYKWR (SEQ IDNO: 118), RYYKWK (SEQ ID NO: 119), RYYRWR (SEQ ID NO: 120), and KYYRWK(SEQ ID NO: 121), wherein the amino acid residues are in the L-formunless otherwise specified. The GCRA peptides described herein can alsobe used in combination therapy with polypeptide conjugate modificationsof the Dooley polypeptide described in WO0198324.

Dosage

Dosage levels of active ingredients in a pharmaceutical composition canalso be varied so as to achieve a transient or sustained concentrationof the compound in a subject, especially in and around the site ofinflammation or disease area, and to result in the desired response. Itis well within the skill of the art to start doses of the compound atlevels lower than required to achieve the desired effect and togradually increase the dosage until the desired effect is achieved. Itwill be understood that the specific dose level for any particularsubject will depend on a variety of factors, including body weight,general health, diet, natural history of disease, route and schedulingof administration, combination with one or more other drugs, andseverity of disease.

An effective dosage of the composition will typically be between about 1μg and about 10 mg per kilogram body weight, preferably between about 10μg to 5 mg of the compound per kilogram body weight. Adjustments indosage will be made using methods that are routine in the art and willbe based upon the particular composition being used and clinicalconsiderations.

The guanylate cyclase receptor agonists used in the methods describedabove may be administered orally, systemically or locally. Dosage formsinclude preparations for inhalation or injection, solutions,suspensions, emulsions, tablets, capsules, topical salves and lotions,transdermal compositions, other known peptide formulations and pegylatedpeptide analogs. Agonists may be administered as either the sole activeagent or in combination with other drugs, e.g., an inhibitor ofcGMP-dependent phosphodiesterase and anti-inflammatory agent. In allcases, additional drugs should be administered at a dosage that istherapeutically effective using the existing art as a guide. Drugs maybe administered in a single composition or sequentially.

Dosage levels of the GCR agonist for use in methods of this inventiontypically are from about 0.001 mg to about 10,000 mg daily, preferablyfrom about 0.005 mg to about 1,000 mg daily. On the basis of mg/kg dailydose, either given in single or divided doses, dosages typically rangefrom about 0.001/75 mg/kg to about 10,000/75 mg/kg, preferably fromabout 0.005/75 mg/kg to about 1,000/75 mg/kg.

The total daily dose of each inhibitor can be administered to thepatient in a single dose, or in multiple subdoses. Typically, subdosescan be administered two to six times per day, preferably two to fourtimes per day, and even more preferably two to three times per day.Doses can be in immediate release form or sustained release formsufficiently effective to obtain the desired control over the medicalcondition.

The dosage regimen to prevent, treat, give relief from, or ameliorate amedical condition or disorder, or to otherwise protect against or treata medical condition with the combinations and compositions of thepresent invention is selected in accordance with a variety of factors.These factors include, but are not limited to, the type, age, weight,sex, diet, and medical condition of the subject, the severity of thedisease, the route of administration, pharmacological considerationssuch as the activity, efficacy, pharmacokinetics and toxicology profilesof the particular inhibitors employed, whether a drug delivery system isutilized, and whether the inhibitors are administered with other activeingredients. Thus, the dosage regimen actually employed may vary widelyand therefore deviate from the preferred dosage regimen set forth above.

EXAMPLES Example 1 Synthesis and Purification of Gcra Peptides

The GCRA peptides were synthesized using standard methods forsolid-phase peptide synthesis. Either a Boc/Bzl or Fmoc/tBu protectinggroup strategy was selected depending upon the scale of the peptide tobe produced. In the case of smaller quantities, it is possible to getthe desired product using an Fmoc/tBu protocol, but for largerquantities (1 g or more), Boc/Bzl is superior.

In each case the GCRA peptide was started by either using a pre-loadedWang (Fmoc) or Merrifield (Boc) or Pam (Boc) resin. For products withC-terminal Leu, Fmoc-Leu-Wang (D-1115) or Boc-Leu-Pam resin (D-1230) orBoc-Leu-Merrifield (D-1030) Thus, for peptides containing the C-terminald-Leu, the resin was Fmoc-dLeu-Wang Resin (D-2535) andBoc-dLeu-Merrifield, Boc-dLeu-Pam-Resin (Bachem Product D-1230 andD-1590, respectively) (SP-332 and related analogs). For peptidesproduced as C-terminal amides, a resin with Ramage linker (BachemProduct D-2200) (Fmoc) or mBHA (Boc) (Bachem Product D-1210 was used andloaded with the C-terminal residue as the first synthetic step.

Fmoc-tBu Overview

Each synthetic cycle consisted deprotection with 20% piperidine in DMF.Resin washes were accomplished with alternating DMF and IpOH to swelland shrink the resin, respectively. Peptide synthesis elongated thechain from the C-terminus to the N-terminus. Activation chemistry foreach amino acid was with HBTU/DIEA in a 4 fold excess for 45 minutes. Inautomated chemistries, each amino acid was double coupled to maximizethe coupling efficiency. To insure the correct position of disulfidebonds, the Cys residues were introduced as Cys(Acm) at positions 15 and7. Cys(Trt) was positioned at Cys4 and Cys12. This protecting groupstrategy yields the correct topoisomer as the dominant product (75:25).(For enterotoxin analogs, a third disulfide bond protecting group (Mob)was utilized).

For peptides containing C-terminal Aeea (aminoethyloxyethyloxyacetyl)groups, these were coupled to a Ramage amide linker using the sameactivation chemistry above by using an Fmoc-protected Aeea derivative.The Cys numbering in these cases remains the same and the positioning ofthe protecting groups as well. For the peptides containing theN-terminal extension of Aeea, the Cys residue numbering will beincreased by three Cys4 becomes Cys7, Cys12 becomes Cys15; Cys7 becomesCys10 and Cys 15 becomes Cys18. The latter pair is protected with Acmand the former pair keeps the Trt groups.

For analogs containing D-amino acid substitutions, these were introduceddirectly by incorporating the correctly protected derivative at thedesired position using the same activation chemistry described in thisdocument. For Fmoc strategies, Fmoc-dAsn(Trt)-OH, Fmoc-dAsn(Xan)-OH,Fmoc-dAsp(tBu)-OH, Fmoc-dGlu(tBu)-OH and for Boc strategies,Boc-dAsn(Xan)-OH, Boc-dAsn(Trt)-OH, Boc-dAsp(Chx), Boc-dAsp(Bzl)-OH,Boc-dGlu(Chx)-OH and Boc-dGlu(Bzl)-OH would be utilized.

Each peptide is cleaved from the solid-phase support using a cleavagecocktail of TFA:H2O:Trisisopropylsilane (8.5:0.75:0.75) ml/g of resinfor 2 hr at RT. The crude deprotected peptide is filtered to remove thespent resin beads and precipitated into ice-cold diethylether.

Each disulfide bonds was introduced orthogonally. Briefly, the crudesynthetic product was dissolved in water containing NH₄OH to increasethe pH to 9. Following complete solubilization of the product, thedisulfide bond was made between the Trt deprotected Cys residues bytitration with H₂O₂. The monocyclic product was purified by RP-HPLC. Thepurified mono-cyclic product was subsequently treated with a solution ofiodine to simultaneously remove the Acm protecting groups and introducethe second disulfide bond.

For enterotoxin analogs, the Mob group was removed via treatment of thedicyclic product with TFA 85% containing 10% DMSO and 5% thioanisole for2 hr at RT.

Each product was then purified by RP-HPLC using a combination buffersystem of TEAP in H2O versus MeCN, followed by TFA in H2O versus MeCN.Highly pure fractions were combined and lyophilized. The final productwas converted to an Acetate salt using either ion exchange with Acetateloaded Dow-Ex resin or using RP-HPLC using a base-wash step with NH₄OAcfollowed by 1% AcOH in water versus MeCN.

It is also possible to prepare enterotoxin analogs using a randomoxidation methodology using Cys(Trt) in Fmoc or Cys(MeB) in Boc.Following cleavage, the disulfide bonds can be formed using disulfideinterchange redox pairs such as glutathione (red/ox) and/orcysteine/cystine. This process will yield a folded product that thedisulfide pairs must be determined as there would be no way of knowingtheir position directly.

Boc-Bzl Process

Peptide synthesis is initiated on a Merrifield or Pam pre-loaded resinor with mBHA for peptides produced as C-terminal amides. Each syntheticcycle consists of a deprotection step with 50% TFA in MeCL2. The resinis washed repetitively with MeCl2 and MeOH. The TFA salt formed isneutralized with a base wash of 10% TEA in MeCl2. The resin is washedwith MeCl2 and MeOH and lastly with DMF prior to coupling steps. Acolorimetric test is conducted to ensure deprotection. Each coupling ismediated with diisopropyl carbodiimide with HOBT to form the activeester. Each coupling is allowed to continue for 2 hr at RT or overnighton difficult couplings. Recouplings are conducted with either Uronium orPhosphonium reagents until a negative colorimetric test is obtained forfree primary amines. The resin is then washed with DMF, MeCl2 and MeOHand prepared for the next solid-phase step. Cys protection utilizesCys(Acm) at positions 7 and 15, and Cys(MeB) at Cys 4 and Cys12.

Cleavage and simultaneous deprotection is accomplished by treatment withHF using anisole as a scavenger (9:1:1) ml:ml:g (resin) at 0° C. for 60min. The peptide is subsequently extracted from the resin andprecipitated in ice cold ether. The introduction of disulfide bonds andpurification follows the exact same protocol described above for theFmoc produced product.

Example 2 In Vitro Proteolytic Stability Using Simulated Gastric Fluid(SGF) Digestion

The stability of SP-304 in the presence of simulated gastric fluid (SGF)was determined. SP-304 (final concentration of 8.5 mg/ml) was incubatedin SGF (Proteose peptone (8.3 g/liter; Difco), D-Glucose (3.5 g/liter;Sigma), NaCl (2.05 g/liter; Sigma), KH₂PO₄ (0.6 g/liter; Sigma), CaCl₂(0.11 g/liter), KCl (0.37 g/liter; Sigma), Porcine bile (final 1×concentration 0.05 g/liter; Sigma) in PBS, Lysozyme (final 1×concentration 0.10 g/liter; Sigma) in PBS, Pepsin (final 1×concentration 0.0133 g/liter; Sigma) in PBS). SGF was made on the day ofthe experiment and the pH was adjusted to 2.0±0.1 using HCl or NaOH asnecessary. After the pH adjustment, SGF is filter sterilized with 0.22μm membrane filters. SP-304 (final concentration of 8.5 mg/ml) wasincubated in SGF at 37° C. for 0, 15, 30, 45, 60 and 120 min,respectively, in triplicate aliquots. Following incubations, sampleswere snap frozen in dry ice then stored in a −80° C. freezer untilassayed in duplicate.

FIG. 1A is a bar chart showing the biological activity of SP-304 afterincubation with SGF for times as indicated. The activity at 0 min wastaken as 100%. The data are an average of triplicates±SD for each datapoint. The data demonstrate that SP-304 is not sensitive to digestionwith SGF. In addition, the data also suggest that the activity of SP-304is not affected by exposure to the acidic pH of the SGF.

These results were further confirmed by the HPLC analyses of the samplesafter digestion with SGF. Here, aliquots of samples from all digestionswere analyzed using a previously developed method for analyzing SP-304peptide using HPLC. Samples from the SGF digestions were diluted to givea final concentration 0.17 mg/mL of SP-304. FIG. 1B shows HPLCchromatographs of SP-304 samples after incubation with SGF at indicatedtimes. The major peak of SP-304 did not change following digestion withSGF, indicating that the peptide was resistant to SGF digestion.

Example 3 In Vitro Proteolytic Stability Using Simulated IntestinalFluid (SIF) Digestion

The stability of SP-304 was also evaluated after incubation withsimulated intestinal fluid (SIF). SIF solution was prepared by themethod as described in the United States Pharmacopoeia, 24th edition,p2236. The recipe to prepare SIF solution was as described below. TheSIF solution contained NaCl (2.05 g/liter; Sigma), KH₂PO₄ (0.6 g/liter;Sigma), CaCl₂ (0.11 g/liter), KCl (0.37 g/liter; Sigma), and Pacreatin10 mg/ml. The pH was adjusted to 6 and the solution was filtersterilized. A solution of SP-304 (8.5 mg/ml) was incubated in SGF at 37°C. for 0, 30, 60, 90, 120, 150 and 300 min respectively, in triplicatealiquots. Following incubations, samples were removed and snap frozenwith dry ice and stored in a −80° C. freezer until assayed in duplicate.FIG. 2A is a bar chart showing the ability of SP-304, after incubationin SIF for times as indicated, to stimulate cGMP synthesis in T84 cells.The cGMP stimulation activity at 0 min was taken as 100%. The data arean average of 3 triplicates±SD. The data indicated that the biologicalactivity of SP-304 is reduced by 30% following digestion with ST. Thiscould be due to degradation of the peptide. Hence, samples afterdigestion with SIF were further analyzed by HPLC.

The integrity of SP-340 peptide exposed to ST was evaluated by HPLC byessentially using the method described for SGF digestion. FIG. 2B is aschematic representation of the results of HPLC chromatographic analysesof SP-304 samples after incubation with heat-inactivated SIF for 300min, and SIF for 120 min, respectively. The major peak of SP-304, whichelutes at 16.2 min was converted into another peak at 9.4 min and a fewminor peptide peaks. Thus, it was important to find out structures ofthe metabolites of SP-304 produced after digestion with SIF. SP-304peptide was incubated with ST for various times and the peptidedigestion products were isolated and subjected to structure elucidationby MS analysis.

FIG. 3 is a schematic representation of the possible metabolites ofSP-304. The major degradation products involve N and D clipped from theN-terminus and L from the C-terminus of SP304. However, there was only30% reduction in biological activity, implying that one or more of thedegradation products were also biologically active. To address thispossibility, several truncated peptides were synthesized and evaluatedfor their abilities to stimulate cGMP synthesis in T84 cells (FIG. 4).

FIG. 4 shows data from the analyses of various peptides in the T84 cellcGMP stimulation assay (essentially as described in Shailubhai, et al.,Cancer Research 60, 5151-5157 (2000). Briefly, confluent monolayers ofT-84 cells in 24-well plates were washed twice with 250 μl of DMEMcontaining 50 mM HEPES (pH 7.4) and pre-incubated at 37° C. for 10minutes with 250 μl of DMEM containing 50 mM HEPES (pH 7.4) and 1 mMisobutyl methylxanthine (IBMX). Monolayers of T84 cells were thenincubated with 250 μl of DMEM containing 50 mM HEPES (pH 7.4) containingone of the peptides shown in the FIG. 4 at a concentration of 1.0 μM for30 min. After the 30 min incubation, the medium was aspirated and thereaction was terminated by the addition of 3% perchloric acid. Followingcentrifugation and the addition of NaOH (0.1 N) to neutralize the pH,intracellular cGMP levels were determined in lysates using a cGMP ELISAkit (Cat. No. 581021; Cayman Chemical, Ann Arbor, Mich.). Samples wererun in duplicates incubations and each sample was run as duplicates inELISA test.

The data suggest that the leucine (L) residue at the C-terminus ofSP-304 contributes to the biological potency of the peptide. Forexample, there was considerable reduction in potency when L was deletedfrom SP-304, as in SP-338. Similarly, the peptides SP-327, SP-329 andSP-331, without L at the C-terminal, also showed 20-25% reduction inbiological potency as compared to their counterpart peptides with L atthe C-terminus, as in SP-326, SP-328 and SP-330 peptides. In addition,results also suggest that amino acid residues at the N-terminus mightalso be important for stability and/or potency of the peptides. Based onthese results, several new peptides were synthesized with D-forms ofamino acids replacing the corresponding L-forms at the C- and N-terminiof the peptides. These peptides were evaluated for their abilities tostimulate cGMP synthesis in T84 cells as shown in FIG. 5.

The results presented in FIG. 5 suggest that substitution of L-aminoacids with D-amino acids at the C- and N-termini did not significantlyalter their potency. Peptides SP-332, SP-333 and SP-335 showedcomparable ability to stimulate cGMP synthesis in T84 cells. On theother hand, the substitution of L-leucine with D-leucine at the 6^(th)position in SP-337 resulted in a complete loss in its ability tostimulate cGMP synthesis in T84 cells. These results suggest that theamino acid residues Asn, Asp and Glu at the N-terminus and Leu at theC-terminus can be replaced with their respective D-amino acid forms.However, the leucine at the 6^(th) position can not be replaced with itsD-form.

FIG. 7 (A-F) shows the stabilities of peptides SP-332, SP-333 and SP-304when incubated with SIF for two hours. The results demonstrated that thepeptide SP-333, which has D-Asn at the N-terminus and D-Leu at theC-terminus, was virtually completely resistant to digestion with SIF(FIG. 7F), and remained virtually 100% biologically active after a twohour incubation in SIF (FIG. 7A). The peptide SP-332 with D-Leu at theC-terminus showed some reduction in potency following the 120 minincubation with SIF (FIG. 7B). However, the HPLC analyses of SP-332 didnot reveal any degradation of the peptide (FIG. 7E), suggesting thatthese peptides are completely resistant to proteoysis by SIF. On theother hand, the peptide SP-304 lost about 30% of its potency followingdigestion with SIF for just one hour (FIG. 7C). The HPLC analysis ofSP-304 following SIF incubation confirmed its degradation (FIG. 7D).These results suggest that the peptide SP-304 undergoes proteolysisfollowing incubation with SIF, whereas substitution of L-Asn with D-Asnat the N-terminus plus the substitution of L-Leu with D-Leu at theC-terminus protects SP-333 against digestion with SIF. Thus, the peptideSP-333 appears more stable and potent as a drug candidate.

Example 4 Cyclic GMP Stimulation Assays

The ability of the GCRA peptide to bind to and activate the intestinalGC-C receptor was tested by usingT 84 human colon carcinoma cell line.Human T84 colon carcinoma cells were obtained from the American TypeCulture Collection. Cells were grown in a 1:1 mixture of Ham's F-12medium and Dulbecco's modified Eagle's medium (DMEM) supplemented with10% fetal bovine serum, 100 U penicillin/ml, and 100 μg/ml streptomycin.The cells were fed fresh medium every third day and split at aconfluence of approximately 80%.

Biological activity of the GCRA peptides was assayed as previouslyreported (Shailubhai, et al., Cancer Research 60, 5151-5157 (2000)).Briefly, the confluent monolayers of T-84 cells in 24-well plates werewashed twice with 250 μl of DMEM containing 50 mM HEPES (pH 7.4),pre-incubated at 37° C. for 10 min with 250 μl of DMEM containing 50 mMHEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX), followed byincubation with GCRA peptides (0.1 nM to 10 .mu.M) for 30 min. Themedium was aspirated, and the reaction was terminated by the addition of3% perchloric acid. Following centrifugation, and neutralization with0.1 N NaOH, the supernatant was used directly for measurements of cGMPusing an ELISA kit (Caymen Chemical, Ann Arbor, Mich.).

FIG. 6 shows results from the experiments evaluating potency of peptidesthat are similar to the E. coli enterotoxin ST peptide in the cGMPstimulation assay (as above). Among these the peptides SP-353 and SP-354were found to be quite potent to stimulate cGMP synthesis in T84 cells.Particularly, the peptide SP-353 that has Ser residue at the 6^(th)position was found to be the most potent among the peptides tested. Thepeptide SP-355 that has D-Tyr at the C-terminus showed potency markedlyless than the other peptides.

Example 5 Peggylated Peptides

An additional strategy to render peptides more resistant towardsdigestion by digestive proteases is to peggylate them at the N- andC-terminus. The peptide SP-333 was peggylated with theaminoethyloxy-ethyloxy-acetic acid (Aeea) group at the C-terminus(SP-347) or at the N-terminus (SP-350) or at both termini (SP-343).Cyclic GMP synthesis in T84 cells was measured by the method asdescribed above.

The peptides SP-347 and SP-350 showed potencies comparable to SP-333 intheir abilities to stimulate cGMP synthesis in T84 cells. However,peptide SP-343 was considerably less potent as compared to the otherpeptides tested. The poor activity of SP-343 might be due to theconsiderable steric hindrance afforded by the large Aeea groups at bothtermini.

Example 6 Combination of Guanylate Cyclase Receptor Agonists withPhosphodiesterase Inhibitors

Regulation of intracellular concentrations of cyclic nucleotides (i.e.,cAMP and cGMP) and thus, signaling via these second messengers, has beengenerally considered to be governed by their rates of production versustheir rates of destruction within cells. Thus, levels of cGMP in tissuesand organs can also be regulated by the levels of expression ofcGMP-specific phosphodiesterases (cGMP-PDE), which are generallyoverexpressed in cancer and inflammatory diseases. Therefore, acombination consisting of an agonist of GC-C with an inhibitor ofcGMP-PDE might produce synergistic effect on levels of cGMP in thetarget tissues and organs.

Sulindac Sulfone (SS) and Zaprinast (ZAP) are two of the knowninhibitors of cGMP-PDE and have been shown to induce apoptosis in cancercells via a cGMP-dependent mechanism. SS and ZAP in combination withSP304 or SP-333 was evaluated to see if these PDE inhibitors had anysynergistic effect on intracellular accumulation of cGMP (FIG. 9-12). Asthe data shows, SS at concentration of 100 μM did not enhanceintracellular accumulation of cGMP. However, the combination SS withSP304 stimulated cGMP production several fold more then the stimulationby SP304 used alone. This synergistic effect on cGMP levels was morepronounced when SP304 were used at 0.1 μM concentration (FIG. 10).Similar observations were made when SP304 or SP333 were used incombination with ZAP (FIG. 10, FIG. 11 and FIG. 12). These resultssuggest that the intracellular levels of cGMP are stabilized because SSinhibits cGMP-PDE that might be responsible for depletion ofintracellular cGMP. Thus, the approach to use a combination of GC-Cagonist with a cGMP-PDE inhibitor is attractive.

For the results shown in FIG. 9, cyclic GMP synthesis in T84 cells wasassessed essentially as described in Shailubhai et al., Cancer Research60, 5151-5157 (2000). Briefly, confluent monolayers of T-84 cells in24-well plates were washed twice with 250 μl of DMEM containing 50 mMHEPES (pH 7.4) and pre-incubated at 37° C. for 10 minutes with 250 μl ofDMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutyl methylxanthine(IBMX). Monolayers of T84 cells were then incubated with 250 μl of DMEMcontaining 50 mM HEPES (pH 7.4) containing SP-304 or PDE inhibitorseither alone or in combinations, as indicated below in the followingexperimental sets: 1) Control; 2) SP-304 (0.1 μM); 3) Sulindac Sulfone(100 μM); 4) Zaprinast (100 μM); 5) SP-304 (0.1 μM)+Sulindac Sulfone(100 μM); and 6) SP-304 (0.1 μM)+Zaprinast (100 μM). After the 30 minincubation, the medium was aspirated and the reaction was terminated bythe addition of 3% perchloric acid. Following centrifugation and theaddition of NaOH (0.1 N) to neutralize the pH, intracellular cGMP levelswere determined in lysates using a cGMP ELISA kit (Cat. No. 581021;Cayman Chemical, Ann Arbor, Mich.). Samples were run in duplicatesincubations and each sample was run as duplicates in ELISA test.

For the results shown in FIG. 10, the method used was same as the oneused for FIG. 9 except that the monolayers of T84 cells were incubatedwith 500 μl of DMEM containing 50 mM HEPES (pH 7.4) containing SP-304(0.1 or 1.0 μM) or increasing concentrations of PDE inhibitors (0 to 750μM) either alone or in combination with SP-304. After the 30 minincubation, the medium was aspirated and the reaction was terminated bythe addition of 3% perchloric acid. Following centrifugation and theaddition of NaOH (0.1 N) to neutralize the pH, intracellular cGMP levelswere determined in lysates using a cGMP ELISA kit (Cat. No. 581021;Cayman Chemical, Ann Arbor, Mich.). Samples were run as triplicates inELISA test.

For the results shown in FIG. 11, the method used was same as the oneused for FIG. 10 except that the monolayers of T84 cells were incubatedwith 500 μl of DMEM containing 50 mM HEPES (pH 7.4) containing SP-3333(0.1 or 1.0 μM) or increasing concentrations of ZAP (0 to 500 μM) eitheralone or in combination with SP-333. After the 30 min incubation, themedium was aspirated and the reaction was terminated by the addition of3% perchloric acid. Following centrifugation and the addition of NaOH(0.1 N) to neutralize the pH, intracellular cGMP levels were determinedin lysates using a cGMP ELISA kit (Cat. No. 581021; Cayman Chemical, AnnArbor, Mich.). Samples were run as triplicates in ELISA test. For theresults shown in FIG. 12, the method used was same as the one used forFIG. 10 except that the monolayers of T84 cells were incubated with 500μl of DMEM containing 50 mM HEPES (pH 7.4) containing SP-333 (0.1 μM) orincreasing concentrations of Sulindac Sulfone (0 to 500 μM) either aloneor in combination with SP-333. After the 30 min incubation, the mediumwas aspirated and the reaction was terminated by the addition of 3%perchloric acid. Following centrifugation and the addition of NaOH (0.1N) to neutralize the pH, intracellular cGMP levels were determined inlysates using a cGMP ELISA kit (Cat. No. 581021; Cayman Chemical, AnnArbor, Mich.). Samples were run as triplicates using the ELISA test.

Example 7 An Oral Range-Finding Toxicity Study in Cynomolgus Monkeys

The objective of the study is to determine the toxicity of the GRCApeptides according to the invention following a single oral gavageadministration to the cynomolgus monkey and to allow assessment ofreversibility of any changes following a minimum 7-dayobservation/washout period. Each GRCA peptide according to the inventionwill be given at two different dose levels.

Experimental Design

The test (e.g., the GRCA peptides according to the invention) andcontrol/vehicle article will be administered in three phases separatedby a minimum 7-day observation period. Each phase will consist of asingle oral gavage administration to female cynomolgus monkeys asindicated in the tables below:

Phase 1:

Eight non-naïve female cynomolgus monkeys will be transferred from theITR Spare

Monkey colony and assigned to four dose groups as follows:

Group Dose Dose Dose Number of Group Desig- Study Level ConcentrationVolume Animals Number nation

(mg/kg) (mg/mL) (mL/kg) (Females) 1 Control/ 1 0 0 10 2 Vehicle

2 Test 1 1 0.1 10 2 Peptides

Following completion of the Phase 1 dosing, all monkeys will be observedfor 33 days. Upon completion of the observation period, all monkeys willbe transferred back to the ITR Spare Monkey Colony.

Phase 2:

The same eight non-naïve female cynomolgus monkeys as previously used inPhase 1 will be transferred from the ITR Spare Monkey colony andassigned to four dose groups as follows:

Group Dose Dose Dose Number of Group Desig- Study Level ConcentrationVolume Animals Number nation Day (mg/kg) (mg/mL) (mL/kg) (Females) 1Control/ 1 10 1 10 2 Vehicle 2 Test 1 10 1 10 2 Peptides

Following completion of the Phase 2 dosing, all monkeys will be observedfor a minimum of 7 days.

Route of Administration

The oral route of administration has been chosen because it is apreferred human therapeutic route.

Preparation of Test and Control/Vehicle Articles

The test and control/vehicle articles will be prepared fresh on the dayof dosing in cold distilled water (maintained in an ice water bath). Asufficient amount of test article powder will be added to theappropriate amount of distilled water in order to achieve the desiredconcentration. The dose formulations will be mixed by simple inversion.

Analysis of Test Article Concentration and Stability in the DoseFormulations

For possible confirmation of the concentration and stability of the testarticle in the formulations, representative samples will be taken fromthe middle of each concentration, including the control/vehicle articleon the first day of dosing of each group, as indicated below. Sampleswill be collected immediately after preparation on Day 1 and again afterdosing is completed on that day and will be stored frozen (approximately80° C. nominal) in 20 mL screw cap vials. Therefore, the remaining doseformulation vials will be returned to the Pharmacy Department as soon aspossible after completion of dosing.

Group 1: 1.5 mL in duplicate from the middle on Day 1 (pre-dose andpost-dose).

Group 2: 1.5 mL in duplicate from the middle on Day 1 (pre-dose andpost-dose).

Group 3: 1.5 mL in duplicate from the middle on Day 1 (pre-dose andpost-dose).

Group 4: 1.5 mL in duplicate from the middle on Day 1 (pre-dose andpost-dose).

The formulations will be maintained cold in an ice water bath during allsampling procedures.

The formulations will be stirred continuously with a stir bar for aminimum of 15 minutes prior to sampling.

The samples will be retained frozen (approximately −80° C. nominal) atITR until requested by the Sponsor to be shipped to a laboratorydesignated by the Sponsor for analysis. The samples can be discardedonce it is determined by the analyst and Study Director that they are nolonger needed. These samples' disposition will be recorded in the rawdata.

If analyzed, a Dose Formulation report will be prepared by the PrincipalInvestigator (Formulation analysis) and will be provided to ITR forinclusion in the final report.

Test System

-   Species/Strain: Cynomolgus Monkey (Macaca Fasicularis)-   Source: orldwide Primates Inc.,    -   P.O. Box 971279    -   Miami, Fla., 33187, USA    -   and    -   Covance Research Products Inc.    -   P.O. Box 549    -   Alice, Tex., 78333, USA-   Total No. of monkeys on study: 8 non-naive females-   Body Weight Range: 2-4 kg at onset of treatment-   Age Range at Start: Young adult at onset of treatment-   Acclimation Period The animals will be transferred from ITR's spare    monkey colony. They are therefore, considered to be fully acclimated    to the laboratory environment.

The actual age and body weight ranges will be noted in the final report.

Administration of the Test and Control/Vehicle Articles

The test and control/vehicle articles will be administered by oralgavage administration using a gavage tube attached to a syringe in threePhases separated by a minimum 7-day observation/washout period. Eachdosing session will consist of a single oral gavage administration. Thegavage tube will be flushed with 3 mL of reverse osmosis waterimmediately following administration of the dose formulation in order toensure that the entire dose volume has been delivered to the animal. Thedose volume will be 10 mL/kg for all animals, including controls. Theactual volume administered to each monkey on Day 1 of each Phase will becalculated using the Day −1 body weights of each Phase.

Dosing formulations will be maintained cold during dose administrationby placing them in an ice water bath.

The dosing formulations must be placed on a stir plate for a minimum of15 minutes prior to the start of dosing and maintained on the stir platethroughout the dosing procedure.

The dosing formulations must be used within 2 hours of preparation.

Clinical Observations

Cage-side clinical signs (ill health, behavioral changes etc.) will berecorded as indicated below except on detailed clinical examinationdays, where the morning cage-side clinical signs will be replaced by adetailed clinical examination (DCE). During regular cage side clinicalsigns and detailed examinations, particular attention will be paid tostools with respect to amount of stools produced, description of stools,etc.

Cage side clinical signs will be performed as follows:

During the pretreatment period and during the 7-day (minimum)observation periods:

Three times per day with a minimum of 3 hours between each occasion.

On the dosing day of Phase 1: pre-dose, 2, 4, 6, 8 and 24 hourspost-dosing

On the dosing day of Phase 2: pre-dose, continuously for the first 4hours post-dose and at 6, 8 and 24 hours post-dosing

On the dosing day of Phase 3: pre-dose, continuously for the first 4hours post-dose and at 6, 8 and 24 hours post-dosing

A detailed clinical examination of each monkey will be performed once atthe time of animal transfer and once weekly thereafter.

Animals whose health status is judged to warrant additional evaluationwill be examined by a Clinical Veterinarian, or a technician workingunder the supervision of the Clinical Veterinarian. Anyveterinarian-recommended treatments will only be performed onceagreement has been obtained from the Study Director. Where possible, theSponsor will be consulted prior to administration of therapeutic drugs.

Body weights will be recorded for all animals once daily from the day oftransfer through to the end of the study.

Food consumption will be recorded for all animals once daily from theday of transfer through to the end of the study.

Cages will be cleaned prior to the start of the daily food consumptionto ensure no food cookies remain in the cage. Monkeys will be fed 7cookies before 12 pm and 7 cookies after 12 pm. The sum of the totalnumber of cookies given for the day will be recorded.

The next morning, a visual check will be performed to see how manycookies are left in the cage. The number of whole cookies remaining inthe food hopper or on the tray will be recorded. The number of wholecookies left will be subtracted from the total number of cookies givenin order to calculate the number of cookies eaten.

Example 8 Suckling Mouse Model of Intestinal Secretion (SuMi Assay)

The GCRA peptides described herein can be tested for their ability toincrease intestinal secretion using a suckling mouse model of intestinalsecretion. In this model a GCRA peptide is administered to suckling micethat are between seven and nine days old. After the mice are sacrificed,the gastrointestinal tract from the stomach to the cecum is dissected(“guts”). The remains (“carcass”) as well as the guts are weighed andthe ratio of guts to carcass weight is calculated. If the ratio is above0.09, one can conclude that the test compound increases intestinalsecretion. Controls for this assay may include wild-type SP-304, STpolypeptide and Zelnorm®. Phenylbenzoquinone-induced writhing model

The PBQ-induced writhing model can be used to assess pain controlactivity of the GCRA peptide described herein. This model is describedby Siegmund et al. (1957 Proc. Soc. Exp. Bio. Med. 95:729-731). Briefly,one hour after oral dosing with a test compound, e.g., a GCRA peptide,morphine or vehicle, 0.02% phenylbenzoquinone (PBQ) solution (12.5mL/kg) is injected by intraperitoneal route into the mouse. The numberof stretches and writhings are recorded from the 5^(th) to the 10^(th)minute after PBQ injection, and can also be counted between the 35^(th)and 40^(th) minute and between the 60^(th) and 65^(th) minute to providea kinetic assessment. The results are expressed as the number ofstretches and writhings (mean±SEM) and the percentage of variation ofthe nociceptive threshold calculated from the mean value of thevehicle-treated group. The statistical significance of any differencesbetween the treated groups and the control group is determined by aDunnett's test using the residual variance after a one-way analysis ofvariance (P<0.05) using SigmaStat Software.

Example 9 Pharmacokinetic Property Determination of Gcra Peptides

Serum samples are extracted from the whole blood of exposed (mice dosedorally or intravenously with GCRA peptides (s) described herein) andcontrol mice, then injected directly (10 mL) onto an in-line solid phaseextraction (SPE) column (Waters Oasis HLB 25 μm column, 2.0×15 mm directconnect) without further processing. The sample on the SPE column iswashed with a 5% methanol, 95% dH₂O solution (2.1 mL/min, 1.0 minute),then loaded onto an 0 analytical column using a valve switch that placesthe SPE column in an inverted flow path onto the analytical column(Waters Xterra MS C8 5 μm IS column, 2.1×20 mm). The sample is elutedfrom the analytical column with a reverse phase gradient (Mobile PhaseA: 10 mM ammonium hydroxide in dH₂O, Mobile Phase B: 10 mM ammoniumhydroxide in 80% acetonitrile and 20% methanol; 20% B for the first 3minutes then ramping to 95% B over 4 min. and holding for 2 5 min., allat a flow rate of 0.4 mL/min.). At 9.1 minutes, the gradient returns tothe initial conditions of 20% B for 1 min. polypeptide is eluted fromthe analytical column and is detected by triple-quadrapole massspectrometry (MRM, 764 (+2 charge state)>182 (+1 charge state) Da; conevoltage=30V; collision=20 eV; parent resolution=2 Da at base peak;daughter resolution=2 Da at base peak). Instrument response is convertedinto concentration units by comparison with a standard curve using knownamounts of chemically synthesized polypeptide(s) prepared and injectedin mouse plasma using the same procedure.

Similarly, pharmacokinetic properties are determined in rats using LCMSmethodology. Rat plasma samples containing the GCRA peptide areextracted using a Waters Oasis MAX 96 well solid phase extraction (SPE)plate. A 200 μL volume of rat plasma is mixed with 200 μL of ¹³Cg,¹⁵N-labeled polypeptide in the well of a prepared SPE plate. The samplesare drawn through the stationary phase with 15 mm Hg vacuum. All samplesare rinsed with 200 μL of 2% ammonium hydroxide in water followed by 200μL of 20% methanol in water. The samples are eluted with consecutive 100μL volumes of May 20, 1975 formic acid/water/methanol and 100 μL May 15,1980 formic acid/water/methanol. The samples are dried under nitrogenand resuspended in 100 μL of 20% methanol in water. Samples are analyzedby a Waters Quattro Micro mass spectrometer coupled to a Waters 1525binary pump with a Waters 2777 autosampler. A 40 μL volume of eachsample is injected onto a Thermo Hypersil GOLD C18 column (2.1×50 mm, 5um). polypeptide is eluted by a gradient over 3 minutes withacetonitrile and water containing 0.05% trifluoroacetic acid. TheQuattro Micro mass spectrometer is run in multiple reaction monitoring(MRM) mode using the mass transitions of, for example 764>182 or682>136. Using this methodology, polypeptide is dosed orally and by IVto rats at 10 mg/kg. Pharmacokinetic properties including area under thecurve and bioavailability are determined.

Example 10 Diuresis Related Experiments Effect on Diuresis andNatriuresis

The effect of GCRA peptides described herein on diuresis and natriuresiscan be determined using methodology similar to that described inWO06/001931 (examples 6 (p. 42) and 8 (p. 45)). Briefly, thepolypeptide/agonist described herein (180-pmol) is infused for 60 mininto a group of 5 anesthetized mice or primates. Given an estimated ratplasma volume of 10 mL, the infusion rate is approximately 3pmol/mL/min. Blood pressure, urine production, and sodium excretion aremonitored for approximately 40 minutes prior to the infusion, during theinfusion, and for approximately 50 minutes after the infusion to measurethe effect of the GCRA peptides on diuresis and natriuresis. Forcomparison, a control group of five rats is infused with regular saline.Urine and sodium excretion can be assessed. Dose response can also bedetermined. polypeptide/GC-C agonist described herein is infusedintravenously into mice or primates over 60 minutes. Urine is collectedat 30 minute intervals up to 180 minutes after termination ofpolypeptide/GC-C agonist infusion, and urine volume, sodium excretion,and potassium excretion are determined for each collection interval.Blood pressure is monitored continuously. For each dose a dose-responserelationship for urine volume, sodium and potassium excretion can bedetermined. Plasma concentration of the polypeptide/GC-agonist is alsodetermined before and after iv infusion.

Mouse or Primate Diuresis Experiment: Once an appropriate level ofanesthesia has been achieved, a sterile polyurethane catheter isinserted into the urethra and secured using 1-2 drops of veterinary bondadhesive applied to urethra/catheter junction. Animals are then dosedwith either vehicle or test article via the intravenous orintraperitoneal route. Animals are allowed to regain consciousness, andthe volume of urine excreted over a 1-5 hour duration is recordedperiodically for each rat.

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We claim:
 1. A peptide consisting essentially of the amino acid sequenceof SEQ ID NO:9, wherein said peptide is a [4:12, 7:15] bicyclic peptide.2. A pharmaceutical composition in unit dose comprising a guanylatecyclase receptor agonist peptide having the sequence of SEQ ID NO:9,wherein said peptide is a [4:12, 7:15] bicyclic peptide, present in atherapeutically effective amount and a pharmaceutical carrier, excipientor diluent.
 3. The pharmaceutical composition of claim 2, wherein theunit dose form is selected from the group consisting of a tablet, acapsule, a solution or inhalation formulation.
 4. A method of increasingcGMP production in a cell comprising contacting said cell with a peptideconsisting of the amino acid sequence of SEQ ID NO:9, wherein saidpeptide is a [4:12, 7:15] bicyclic peptide.
 5. The method of claim 4,further comprising contacting said cell with a cGMP-dependentphosphodiesterase inhibitor.
 6. The method of claim 5, wherein saidcGMP-dependent phosphodiesterase inhibitor is selected from the groupconsisting of sulindac sulfone, zaprinast, and motapizone, vardenafil,and sildenafil.